Rotavirus-like particle production in plants

ABSTRACT

A method of producing a rotavirus-like particle (RLP) in a plant is provided. The method comprises expressing within a host or host cell for example a plant, portion of a plant or plant cell one or more nucleic acid comprising one or more regulatory region operatively linked to a first, second and third nucleotide sequence, the regulatory region active in the host or host cell. The first nucleotide sequence encoding a first rotavirus protein, the second nucleotide sequence encoding a second rotavirus protein and the third nucleotide sequence encoding a third rotavirus protein. The first, second and third encode rotavirus protein NSP4 and VP2 or VP6 and VP4 or VP7. The host or host cell is incubated under conditions that permit the expression of the nucleic acids, so that NSP4 and either VP2 of VP6 and VP4 or VP7 are expressed, thereby producing the RLP. Hosts comprising the RLP, compositions comprising the RLP and method for using the composition are also provided.

FIELD OF INVENTION

This invention relates to producing rotavirus-like particles in plants.

BACKGROUND OF THE INVENTION

Rotavirus infection is a global problem mainly affecting children underthe age of five. It results in severe gastroenteritis and in worst casesdeath.

Rotaviruses are members of the Reoviridae family of viruses (genusRotavirus) that affect the gastrointestinal system and respiratorytract. The name is derived from the wheel like appearance of virionswhen viewed by negative contrast electron microscopy. The rotavirus isusually globular shape and is named after the outer and inner shells ordouble-shelled capsid structure of the same. The outer capsid is about70 nm, and inner capsid is about 55 nm in diameter, respectively. Thedouble-shelled capsid of the rotavirus surrounds the core including theinner protein shell and genome. The genome of the rotavirus consists ofdouble stranded RNA segments encoding at least 11 rotavirusproteins—either structural viral proteins (VP) or nonstructural proteins(NSP; Desselberger, Virus Res 190: 75-96 (2014)).

The dsRNA codes for six structural proteins (VP) and six non-structuralproteins (NSP). The structural proteins comprise VP1, VP2, VP3, VP4, VP6and VP7. Three concentric layers are formed by the assembly of VP2, VP6and VP7 respectively, with VP4 forming “spikes” on the surface of thevirus structure. VP4 is cleaved by trypsin to VP8* and VP5*. VP8* andVP5* are proteolytic products of VP4.

VP2 is a 102 kDa protein and is the most abundant protein of the viralcore. It forms the inner-most structural protein layer and provides ascaffold for the correct assembly of the components and transcriptionenzymes of the viral core (Lawton, 2000). VP1, the largest viral proteinat 125 kDa, acts as an RNA-dependent polymerase for rotavirus, creatinga core replication intermediate, and associates with VP2 at itsicosahedral vertices (Varani and Allain, 2002; Vende et al., 2002). VP3,a 98 kDa protein, is also directly associated with the viral genome,acting as an mRNA capping enzyme that adds a 5′ cap structure to viralmRNAs. Together, VP1 and VP3 form a complex that is attached to theouter 5-fold vertices of the VP2 capsid layer (Angel, 2007). VP6 is a 42kDa protein which forms the middle shell of the viral core, is the majorcapsid protein and accounts for more than 50% of the total protein massof the virion (González et al., 2004; Estes, 1996). It is required forgene transcription and may have a role in encapsulation of the rotavirusRNA by anchoring VP1 to VP2 in the core, as seen in bluetongue virus,another member of the Reoviridae family. It also determines theclassification of rotaviruses into five groups (A to E) with group Amost commonly affecting humans (Palombo, 1999). VP6 in rotavirus group Ahas at least four subgroups (SG), which depend on the presence orabsence of SG specific epitopes: SG I, SG II, SG (I+II) and SGnon-(I+II). Groups B and C lack a common group A antigen but are alsoknown to infect humans, while group D only affects animals e.g. chickensand cows (Thongprachum, 2010).

The two outer capsid proteins VP7, a 37 kDa glycoprotein (G) and the 87kDa protease sensitive VP4 (P), define the virus' serotypes. These twoproteins induce neutralizing antibody responses and are thus used toclassify rotavirus serotypes into a dual nomenclature system, dependingon the G-P antigen combination (e.g. G1 P[8] or G2 P[4])(Sanchez-Padilla et al., 2009, Rahman et al., J Clin Microbiol 41:2088-2095 (2003)). The VP4 protein dimerizes to form 60 spikes on theouter shell of the virus, which are directly involved in the initialstages of host cell entry. The spike protein contains a cleavage site atamino acid (aa) position 248. Upon infection, it is cleaved by theprotease trypsin to produce VP5 (529 aa, 60 kDa) and VP8 (246 aa, 28kDa) (Denisova et al., 1999). This process enhances virus infectivity(cell attachment and invasion of host cell) and stabilizes the spikestructure (Glass, 2006). The VP7 glycoprotein forms the third or outsidelayer of the virus. At present, 27 G and 35 P genotypes are known(Greenberg and Estes, 2009). VP4 and VP7 are the major antigens involvedin virus neutralization and are important targets for vaccinedevelopment (Dennehy, 2007).

The non-structural proteins (NSPs) are synthesized in infected cells andfunction in various parts of the replication cycle or interact with someof the host proteins to influence pathogenesis or the immune response toinfection (Greenberg and Estes, 2009). The rotavirus nonstructuralprotein, NSP4, has been shown to have multiple functions including therelease of calcium from the endoplasmic reticulum (ER; Tian et al,1995); the disruption of the ER membranes and may play an important rolein the removal of the transient envelope from budding particles duringviral morphogenesis (see FIG. 1); affecting membrane trafficking fromthe ER to the Golgi complex with its ability to bind to micro tubules(Xu et al 2000); and function as an intracellular receptor to aid in thebudding of subviral particles into the ER (Tian et al 1996).

In infected mammalian cells, rotaviruses undergo a unique mode ofmorphogenesis to form the complete triple-layered VP2/6/4/7 viralparticles (Lopez et al., 2005). The triple-layer capsid is a very stablecomplex which enables faecal-oral transmission and delivery of the virusinto the small intestine where it infects non-dividing differentiatedenterocytes near the tips of the villi (Greenberg and Estes, 2009).Firstly, the intact virus attaches to sialic acid-independent receptorsvia 60 VP4 dimer spikes on the surface of the virus (Lundgren andSvensson, 2001). The 60 VP4 dimer spikes on the surface of the virusallow the virus to attach to these cell receptors. VP4 is susceptible toproteolytic cleavage by trypsin which results in a conformational changethat exposes additional attachment sites on the surface of theglycoprotein for interaction with a series of co-receptors.

The multi-step attachment and entry process is, however, not clearlyunderstood but the virus is delivered across the host's plasma membrane.The VP7 outer capsid shell which is also involved in the entry process,is removed in the process and double-layered particles (DLP) aredelivered into the cell cytoplasm in vesicles (FIG. 1; prior art). TheDLP escapes from the vesicle and goes into non-membrane boundcytoplasmic inclusions. Early transcription of the genome by VP1 beginsin particles so that dsRNA is never exposed to the cytoplasm. RNAreplication and core formation takes place in these non-membrane-boundcytoplasmic inclusions. The nascent (+) RNAs are then transported intothe cytoplasm and serve as templates for viral protein synthesis. VP4 isproduced in the cytosol and transported to the rough endoplasmicreticulum (RER), and VP7 is secreted into the RER. VP2 and VP6 areproduced and assemble in the cytosol in virosomes and subsequently budinto the RER compartments, receiving a transient membrane envelope inthe process (Lopez et al., 2005; Tian et al., 1996). In the RER, thetransient envelopes of the viral particles are removed and replaced byVP4 and VP7 protein monomers, with critical involvement of rotaviralglycoprotein NSP4 (Tian et al., 1996; Lopez et al., 2005; Gonzalez etal., 2000). NSP4 functions as an intracellular receptor in the ERmembrane and binds newly made subviral particles and probably also thespike protein VP4 (Tian et al., 1996). NSP4 is also toxic to humans andis the causative agent of the diarrhea. The complete, mature particlesare subsequently transferred from the RER through the Golgi apparatus tothe plasma membrane for secretion (Lopez et al., 2005).

A variety of different approaches have been taken to generate arotavirus vaccine suitable to protect human populations from the variousserotypes of rotavirus. These approaches include various Jennerianapproaches, use of live attenuated viruses, use of virus-like particles,nucleic acid vaccines and viral sub-units as immunogens. At presentthere are two oral vaccines available on the market, however, these havelow efficacy in due to strain variation.

U.S. Pat. Nos. 4,624,850, 4,636,385, 4,704,275, 4,751,080, 4,927,628,5,474,773, and 5,695,767, each describe a variety of rotavirus vaccinesand/or methods of preparing these vaccines, where the whole viralparticles is used to create each of the rotavirus vaccines.

Production of rotavirus-like particles is a challenging task, as boththe synthesis and assembly of one or more recombinant proteins arerequired. Rotavirus comprises a capsid formed by 1860 monomers of fourdifferent proteins. For RLP production the simultaneous expression andassembly of two to three recombinant proteins may be required. Forexample, an inner layer comprising 120 molecules of VP2, 780 moleculesof VP6 (middle layer) and an outer layer of 780 molecules of theglycoprotein VP7 and 60 VP4 dimers, to form a double or triple-layeredparticle (Libersou et al. J. of Virology, March 2008).

Crawford et al. (J Virol. 1994 September; 68(9): 5945-5952) describe theexpression of VP2, VP4, VP6, and VP7 in a baculovirus expression system.Co-expression of different combinations of the rotavirus majorstructural proteins resulted in the formation of stable virus-likeparticles (VLPs). The co-expression of VP2 and VP6 alone or with VP4resulted in the production of VP2/6 or VP2/4/6 VLPs, which were similarto double-layered rotavirus particles. Co-expression of VP2, VP6, andVP7, with or without VP4, produced triple-layered VP2/6/7 or VP2/4/6/7VLPs, which were similar to native infectious rotavirus particles. TheVLPs maintained the structural and functional characteristics of nativeparticles, as determined by electron microscopic examination of theparticles, the presence of non-neutralizing and neutralizing epitopes onVP4 and VP7, and hemagglutination activity of the VP2/4/6/7 VLPs.

Vaccine candidates generated from rotavirus-like particles of differentprotein compositions have shown potential as subunit vaccines. O'Neal etal. (J. Virology, 1997, 71(11):8707-8717) show that VLPs containing VP 2and VP6, or VP2, VP6, and VP7, and administered to mice with and withoutthe addition of cholera toxin induced protective immunity in immunizedmice. Core-like particles (CLP) and VLPs have also been used to immunizecows with VLPs more effective than CLPs in inducing passive immunityFernandez, et al., (Vaccine, 1998, 16(5):507-516).

Plants are increasingly being used for large-scale production ofrecombinant proteins. For example US 2003/0175303 discloses theexpression of recombinant rotavirus structural protein VP6, VP2, VP4 orVP7 in stably transformed tomato plants.

Saldana et al. (Viral Immunol. 19: 42-53 (2006)) expressed VP2 and VP6in the cytoplasm of tomato plants. Electron microscopy studies showedthat a small proportion of the proteins had assembled into 2/6 VLPs. Aprotective immune response was detected in mice and this may have tosome extent been contributed by the non-assembled VPs. Individualproteins have been shown to elicit immune responses in mice, as in thecase of VP8 and VP6 (Rodriguez-Diaz et al. Biotechnol Lett. 2011,33(6):1169-75, Zhou et al., Vaccine 28: 6021-6027 (2010)).

Matsumura et al., (Archives of Virology 147: 1263-1270 (2002)) reportbovine rotavirus A VP6 expression in transgenic potato plants. The VP6was expressed, purified and immunogenic studies performedImmune-response in adult mice showed presence of VP6 antibodies in thesera. However, no evidence of assembled VP6 proteins was provided. Itmay have been that monomers or trimers of VP6 were responsible foreliciting the immune response. O'Brien et al. (2000, Virol. 270:10444-10453) show VP6 assembly in Nicotiana benthamiana using a potatovirus X (PVX) vector. Assembly of VP6 protein into icosahedral VLPs wasonly observed when the VP6 was fused to the PVX protein rods. Followingcleavage the VP6 assembled into the icosahedral VLPs.

Codon-optimized human rotavirus VP6 has been successfully expressed inChenopodium amaranticolor using a Beet black scorch virus (BBSV)mediated expression system. The protein was engineered as a replacementto the coat protein of BBSV. Oral immunization of female BALB/c micewith the plant based VP6 protein induced high titers of anti-VP6 mucosalIgA and serum IgG (Zhou et al., Vaccine 28: 6021-6027 (2010)). However,there was no teaching that the VP6 proteins assembled into VLPs orparticles.

Rotavirus VP7 has been expressed in potato plants and was shown toproduce a neutralizing immune response in mice (Yu and Langridge, 2001Nature Biotechnol 19: 548-552). In transgenic potato plants, the VP7gene was stable over 50 generations, with the VP7 protein from the 50thgeneration induced both protective and neutralizing antibodies in adultmice (Li et al., 2006, Virol 356:171-178).

Yang et al. (Yang Y M, Li X, Yang H, et al. Science China Life Science54: 82-89 (2011)) co-expressed three rotavirus capsid proteins VP2, VP6and VP7 of group A RV (P[8]G1) in tobacco plants and expression levelsof these proteins, as well as formation of rotavirus-like particles andimmunogenicity were studied. VLPs were purified from transgenic tobaccoplants and analyzed by electron microscopy and Western blot. Theseresults indicate that the plant derived VP2, VP6 and VP7 proteinself-assembled into 2/6 or 2/6/7 rotavirus like particle with a diameterof 60-80 nm.

WO 2013/166609 described the production of rotavirus-like particle(RLPs) in plants, by co-expressing rotavirus structural proteins VP2,VP4, VP6 and VP7 in plants and purifying the resulting RLPs in thepresence of calcium.

Rotavirus NSP4 has been expressed and purified from insect cells (Tianet al. 1996, Arch Virol. 1996; Rodriguez-Diaz et al. Protein Expr.Purif. 2003) and in E. coli (Sharif et al. Medical Journal of theIslamic Republic of Iran 2003). NSP4 has also been expressed as a fusionprotein with the cholera toxin B (CTB) subunit in potato (Arakawa etal., Plant Cell Report 20: 343-348 (2001)).

SUMMARY OF THE INVENTION

The present invention relates to producing rotavirus-like particles inplants.

It is an object of the invention to produce rotavirus-like particles inplants.

Several methods to produce a rotavirus like particle (RLP) in a plant,portion of a plant or plant cell are described.

For example, a method (A) for producing a rotavirus like particle (RLP)in a host or host cell may comprise:

-   a) providing a host or host cell comprising one or more nucleic acid    comprising a first nucleotide sequence encoding a first rotavirus    protein, a second nucleotide sequence encoding a second rotavirus    protein and a third nucleotide sequence encoding a third rotavirus    protein, the first, second and third nucleotide sequence being    operatively linked to one or more regulatory region active in the    host or host cell; and-   the first nucleotide sequence encoding rotavirus protein NSP4, the    second nucleotide sequence encoding rotavirus protein VP6, and the    third nucleotide sequence encoding one of rotavirus protein VP7 or    VP4;-   b) incubating the host or host cell under conditions that permit the    expression of the one or more nucleic acid, so that each of NSP4,    VP6 and VP7 or VP4 are expressed, thereby producing the RLP.

In the method (A) as described above the one or more nucleic acid mayfurther comprises a fourth nucleotide sequence encoding a fourthrotavirus protein, the first, second, third, and fourth nucleotidesequence being operatively linked to one or more regulatory regionactive in the host or host cell; and

-   the first nucleotide sequence encoding rotavirus protein NSP4, the    second nucleotide sequence encoding rotavirus protein VP6, and the    third and fourth nucleotide sequence encoding rotavirus protein VP2,    VP4 or VP7 and wherein each of NSP4, VP6 and two of VP2, VP4 and VP7    are expressed from the one or more nucleic acid.

A method (B) to produce a rotavirus like particle (RLP) in a host orhost cell is further described, the method may comprise:

-   a) providing a host or host cell comprising one or more nucleic acid    comprising-   a first nucleotide sequence encoding a first rotavirus protein, a    second nucleotide sequence encoding a second rotavirus protein and a    third nucleotide sequence encoding a third rotavirus protein, the    first, second and third nucleotide sequence being operatively linked    to one or more regulatory region active in the host or host cell;    and-   the first, second and third nucleotide sequence encoding one of    rotavirus protein NSP4, VP6 and one of rotavirus protein VP7 or VP4;-   b) incubating the host or host cell under conditions that permit the    expression of the one or more nucleic acid, so that each of NSP4,    VP6 and VP7 or VP4 are expressed, thereby producing the RLP.

In the method (B) as described above the one or more nucleic acid mayfurther comprises a fourth nucleotide sequence encoding a fourthrotavirus protein, the first, second, third, and fourth nucleotidesequence being operatively linked to one or more regulatory regionactive in the host or host cell; and

-   the first, second, third and fourth nucleotide sequence encoding one    of rotavirus protein NSP4, VP6 and two of rotavirus protein VP2, VP7    or VP4 and wherein each of NSP4, VP6 and two of VP2, VP7 or VP4 are    expressed from the one or more nucleic acid.

In the method (A) and (B) as described above the one or more nucleicacid may further comprises a fourth nucleotide sequence encoding afourth rotavirus protein and a fifth nucleotide sequence encoding afifth rotavirus protein, the first, second, third, fourth and fifthnucleotide sequence being operatively linked to one or more regulatoryregion active in the host or host cell; and

-   the first, second, third, fourth and fifth nucleotide sequence    encoding one of rotavirus protein VP2, VP4, VP6, VP7 or NSP4 and    wherein each of VP2, VP4, VP6, VP7 and NSP4 are expressed from the    one or more nucleic acid.

In the method (A) or (B) as described above, if a host or host cell isprovided where the one or more nucleic acid comprising a firstnucleotide sequence encoding a first rotavirus protein, a secondnucleotide sequence encoding a second rotavirus protein, a thirdnucleotide sequence encoding a third rotavirus protein, fourthnucleotide sequence encoding a fourth rotavirus protein and a fifthnucleotide sequence encoding a fifth rotavirus protein, the first,second, third, fourth and fifth nucleotide sequence being operativelylinked to one or more regulatory region active in the host or host cell,then the one or more nucleic acid may comprise the first, second, third,fourth and fifth nucleotide sequence encoding the first, second, third,fourth and fifth rotavirus protein, or the one or more nucleic acid maycomprise for example two nucleic acids, a first nucleic acid comprisingthe first nucleotide sequence encoding the first rotavirus protein, anda second nucleic acid comprising the second, third, fourth and fifthnucleotide sequence encoding the second, third, fourth and fifthrotavirus protein, or the one or more nucleic acid may comprise forexample two nucleic acids, a first nucleic acid comprising the first andsecond nucleotide sequence encoding the first and second rotavirusprotein, and a second nucleic acid comprising the third, fourth andfifth nucleotide sequence encoding the third, fourth and fifth rotavirusprotein or the one or more nucleic acid may comprise for example threenucleic acids, a first nucleic acid comprising the first and secondnucleotide sequence encoding the first and second rotavirus protein, asecond nucleic acid comprising the third and fourth nucleotide sequenceencoding the third and fourth rotavirus protein, and a third nucleicacid comprising the fifth nucleotide sequence encoding the fifthrotavirus protein or the one or more nucleic acid may comprise forexample three nucleic acids, a first nucleic acid comprising the firstnucleotide sequence encoding the first rotavirus protein, a secondnucleic acid comprising the second nucleotide sequence encoding thesecond rotavirus protein, and a third nucleic acid comprising the third,fourth and fifth nucleotide sequence encoding the third, fourth andfifth rotavirus protein or the one or more nucleic acid may comprise forexample four nucleic acids, a first nucleic acid comprising the firstnucleotide sequence encoding the first rotavirus protein, a secondnucleic acid comprising the second nucleotide sequence encoding thesecond rotavirus protein, a third nucleic acid comprising the thirdnucleotide sequence encoding the third rotavirus protein, and a fourthnucleic acid comprising the fourth and fifth nucleotide sequenceencoding the fourth and fifth rotavirus protein or the one or morenucleic acid may comprise for example five nucleic acids, a firstnucleic acid comprising the first nucleotide sequence encoding the firstrotavirus protein, a second nucleic acid comprising the secondnucleotide sequence encoding the second rotavirus protein, a thirdnucleic acid comprising the third nucleotide sequence encoding the thirdrotavirus protein, a fourth nucleic acid comprising the fourthnucleotide sequence encoding the fourth rotavirus protein, and a fifthnucleic acid comprising the fifth nucleotide sequence encoding the fifthrotavirus protein.

The methods (A) or (B) as described above may further comprise the stepsof:

-   -   c) harvesting the host or host cell, and    -   d) purifying the RLPs from the host or host cell, wherein the        RLPs range in size from 70-100 nm.

The one or more nucleotide sequence of the method (A) or (B) asdescribed above may be operatively linked to one or more expressionenhancer. Furthermore, the expression enhancer may be selected from thegroup consisting of CPMV HT, CPMV 160, CPMV 160+ and CPMV HT+.

Also described herein is a method (C) of producing a rotavirus likeparticle (RLP) in host or host cell comprising:

-   a) introducing into the host or host cell one or more nucleic acid    comprising-   a first nucleotide sequence encoding a first rotavirus protein, a    second nucleotide sequence encoding a second rotavirus protein and a    third nucleotide sequence encoding a third rotavirus protein, the    first, second and third nucleotide sequence being operatively linked    to one or more regulatory region active in the host or host cell;    and-   the first nucleotide sequence encoding rotavirus protein NSP4, the    second nucleotide sequence encoding rotavirus protein VP6, and the    third nucleotide sequence encoding one of rotavirus protein VP7 or    VP4;-   b) incubating the host or host cell under conditions that permit the    expression of the one or more nucleic acid so that each of NSP4, VP6    and VP7 or VP4 are expressed, thereby producing the RLP.

In the method (C) as described above the one or more nucleic acid mayfurther comprises a fourth nucleotide sequence encoding a fourthrotavirus protein, the first, second, third, and fourth nucleotidesequence being operatively linked to one or more regulatory regionactive in the host or host cell; and

-   the first nucleotide sequence encoding rotavirus protein NSP4, the    second nucleotide sequence encoding rotavirus protein VP6, and the    third and fourth nucleotide sequence encoding rotavirus protein VP2,    VP4 or VP7 and wherein each of NSP4, VP6 and two of VP2, VP4 and VP7    are expressed from the one or more nucleic acid.

Also described herein is a method (D) of producing a rotavirus likeparticle (RLP) in host or host cell comprising:

-   a) introducing into the host or host cell one or more nucleic acid    comprising-   a first nucleotide sequence encoding a first rotavirus protein, a    second nucleotide sequence encoding a second rotavirus protein and a    third nucleotide sequence encoding a third rotavirus protein, the    first, second and third nucleotide sequence being operatively linked    to one or more regulatory region active in the host or host cell;    and-   the first, second and third nucleotide sequence encoding one of    rotavirus protein NSP4, VP6 and one of rotavirus protein VP7 or VP4;-   b) incubating the host or host cell under conditions that permit the    expression of the one or more nucleic acid so that each of NSP4, VP6    and VP7 or VP4 are expressed, thereby producing the RLP.

In the method (D) as described above the one or more nucleic acid mayfurther comprises a fourth nucleotide sequence encoding a fourthrotavirus protein, the first, second, third, and fourth nucleotidesequence being operatively linked to one or more regulatory regionactive in the host or host cell; and

-   the first, second, third and fourth nucleotide sequence encoding one    of rotavirus protein NSP4, VP6 and two of rotavirus protein VP2, VP7    or VP4 and wherein each of NSP4, VP6 and two of VP2, VP7 or VP4 are    expressed from the one or more nucleic acid.

In the method (C) and (D) as described above the one or more nucleicacid may further comprises a fourth nucleotide sequence encoding afourth rotavirus protein and a fifth nucleotide sequence encoding afifth rotavirus protein, the first, second, third, fourth and fifthnucleotide sequence being operatively linked to one or more regulatoryregion active in the host or host cell; and

-   the first, second, third, fourth and fifth nucleotide sequence    encoding one of rotavirus protein VP2, VP4, VP6, VP7 or NSP4 and    wherein each of VP2, VP4, VP6, VP7 and NSP4 are expressed from the    one or more nucleic acid.

The methods (C) or (D) as described above may further comprise the stepsof:

-   c) harvesting the host or host cell, and-   d) purifying the RLPs from the host or host cell, wherein the RLPs    range in size from 70-100 nm.

In the method (C) or (D) as described above, if a host or host cell isprovided where the one or more nucleic acid comprising a firstnucleotide sequence encoding a first rotavirus protein, a secondnucleotide sequence encoding a second rotavirus protein, a thirdnucleotide sequence encoding a third rotavirus protein, fourthnucleotide sequence encoding a fourth rotavirus protein and a fifthnucleotide sequence encoding a fifth rotavirus protein, the first,second, third, fourth and fifth nucleotide sequence being operativelylinked to one or more regulatory region active in the host or host cell,then in the step of introducing (step a), the one or more nucleic acidmay comprise two nucleic acids, with a first nucleic acid comprisingencoding the NSP4, and a second nucleic acid encoding VP2, VP4, VP6 andVP7, the ratio of an amount of the first nucleic acid relative to thesecond nucleic acid that is introduced into the plant, portion of aplant or plant cell is between 1:0.8 and 1:2. The ratio may also be 1:1.Alternatively, the one or more nucleic acid may comprise two nucleicacids, with a first nucleic acid comprising the first nucleotidesequence encoding the first rotavirus protein and the second nucleotidesequence encoding the second rotavirus protein, and a second nucleicacid comprising the third to fifth nucleotide sequence encoding thethird to fifth rotavirus protein.

In the method (C) or (D) as described above, in the step of introducing(step a), the one or more nucleic acid may comprise three nucleic acids,with a first nucleic acid comprising the first and the second nucleotidesequence encoding the first and the second rotavirus protein, a secondnucleic acid comprising the third and fourth nucleotide sequenceencoding the third and the fourth rotavirus protein, and a third nucleicacid comprising the fifth nucleotide sequence encoding the fifthrotavirus protein, and wherein the ratio of an amount of the firstnucleic acid relative to the second nucleic acid and to the thirdnucleic that is introduced into the plant, the portion of the plant, orthe plant cell, is 1:1:1.

Alternatively, in the method (C) or (D) as described above, in the stepof introducing (step a), the one or more nucleic acid may comprise threenucleic acids, with the first nucleic acid comprising the firstnucleotide sequence encoding the first rotavirus protein, the secondnucleic acid comprising the second nucleotide sequence encoding thesecond rotavirus protein, and the third nucleic acid comprising thethird to fifth nucleotide sequence encoding the third to fifth rotavirusprotein, and wherein the ratio of an amount of the first nucleic acidrelative to the second nucleic acid and to the third nucleic that isintroduced into the plant, the portion of the plant, or the plant cell,is 1:1:1.

Furthermore, in method (C) or (D) described above, in the step ofintroducing (step a), the one or more nucleic acid may comprise fournucleic acids, a first nucleic acid comprising the first nucleic acidencoding the first rotavirus protein, a second nucleic acid comprisingthe second nucleotide sequence encoding the second rotavirus protein, athird nucleic acid comprising the third nucleotide sequence encoding thethird rotavirus protein, and a fourth nucleic acid comprising the fourthand the fifth nucleotide sequence encoding the fourth and fifthrotavirus protein, wherein the ratio of an amount of the first nucleicacid relative to the second nucleic acid, to the third nucleic and tothe fourth nucleic acid that is introduced into the plant, the portionof the plant, or the plant cell, is 1:1:1:1.

In the method (C) or (D) as described above, in the step of introducing(step a), the one or more nucleic acid may comprise five nucleic acids,a first nucleic acid comprising the first nucleotide sequence encodingthe first rotavirus protein, a second nucleic acid comprising the secondnucleotide sequence encoding the second rotavirus protein, a thirdnucleic acid comprising the third nucleotide sequence encoding the thirdrotavirus protein, a fourth nucleic acid comprising the fourthnucleotide sequence encoding the fourth rotavirus protein, and a fifthnucleic acid comprising the fifth nucleotide sequence encoding the fifthrotavirus protein, and the ratio of an amount of the first nucleic acidrelative to the second nucleic acid, to the third nucleic acid, to thefourth nucleic acid and to the fifth nucleic acid that is introducedinto the plant, the portion of the plant, or the plant cell, is1:1:1:1:1.

The one or more nucleotide sequence of the method (C) or (D) asdescribed above may be operatively linked to one or more expressionenhancer. Furthermore, the expression enhancer may be selected from thegroup consisting of CPMV HT, CPMV 160, CPMV 160+ and CPMV HT+.

A method (E) of increasing incorporation of VP4, VP7, or both VP4 andVP7 in a rotavirus like particle (RLP) is also described, the methodcomprising:

-   -   a) providing a host or host cell comprising one or more nucleic        acid comprising    -   a first nucleotide sequence encoding a first rotavirus protein,        a second nucleotide sequence encoding a second rotavirus protein        and a third nucleotide sequence encoding a third rotavirus        protein, the first, second and third nucleotide sequence being        operatively linked to one or more regulatory region active in        the host or host cell; and    -   the first nucleotide sequence encoding rotavirus protein NSP4,        the second nucleotide sequence encoding rotavirus protein VP6,        and the third nucleotide sequence encoding one of rotavirus        protein VP7 or VP4;    -   b) incubating the host or host cell under conditions that permit        the expression of the one or more nucleic acid, so that each of        NSP4, VP6 and VP7 or VP4 are expressed, thereby producing the        RLP with enhanced levels of VP4, VP7, or both VP4 and VP7 when        compared to the level of VP4 or VP7 produced by a second host or        host cell that expresses the one or more nucleic acid that does        not comprise NSP4, under the same conditions.

In the method (E) as described above the one or more nucleic acid mayfurther comprises a fourth nucleotide sequence encoding a fourthrotavirus protein, the first, second, third, and fourth nucleotidesequence being operatively linked to one or more regulatory regionactive in the host or host cell; and

-   the first nucleotide sequence encoding rotavirus protein NSP4, the    second nucleotide sequence encoding rotavirus protein VP6, and the    third and fourth nucleotide sequence encoding rotavirus protein VP2,    VP4 or VP7 and wherein each of NSP4, VP6 and two of VP2, VP4 and VP7    are expressed from the one or more nucleic acid.

A method (F) of increasing incorporation of VP4, VP7, or both VP4 andVP7 in a rotavirus like particle (RLP) is also described, the methodcomprising:

-   -   a) providing a host or host cell comprising one or more nucleic        acid comprising

-   a first nucleotide sequence encoding a first rotavirus protein, a    second nucleotide sequence encoding a second rotavirus protein and a    third nucleotide sequence encoding a third rotavirus protein, the    first, second and third nucleotide sequence being operatively linked    to one or more regulatory region active in the host or host cell;    and

-   the first, second and third nucleotide sequence encoding one of    rotavirus protein NSP4, VP6 and one of rotavirus protein VP7 or VP4;    -   b) incubating the host or host cell under conditions that permit        the expression of the one or more nucleic acid, so that each of        NSP4, VP6 and VP7 or VP4 are expressed, thereby producing the        RLP with enhanced levels of VP4, VP7, or both VP4 and VP7 when        compared to the level of VP4 or VP7 produced by a second host or        host cell that expresses the one or more nucleic acid that does        not comprise NSP4, under the same conditions.

In the method (F) as described above the one or more nucleic acid mayfurther comprises a fourth nucleotide sequence encoding a fourthrotavirus protein, the first, second, third, and fourth nucleotidesequence being operatively linked to one or more regulatory regionactive in the host or host cell; and

-   the first, second, third and fourth nucleotide sequence encoding one    of rotavirus protein NSP4, VP6 and two of rotavirus protein VP2, VP7    or VP4 and wherein each of NSP4, VP6 and two of VP2, VP7 or VP4 are    expressed from the one or more nucleic acid.

In the method (E) and (F) as described above the one or more nucleicacid may further comprises a fourth nucleotide sequence encoding afourth rotavirus protein and a fifth nucleotide sequence encoding afifth rotavirus protein, the first, second, third, fourth and fifthnucleotide sequence being operatively linked to one or more regulatoryregion active in the host or host cell; and

-   the first, second, third, fourth and fifth nucleotide sequence    encoding one of rotavirus protein VP2, VP4, VP6, VP7 or NSP4 and    wherein each of VP2, VP4, VP6, VP7 and NSP4 are expressed from the    one or more nucleic acid.

The one or more nucleotide sequence of the method (E) or (F) asdescribed above may be operatively linked to one or more expressionenhancer. Furthermore, the expression enhancer may be selected from thegroup consisting of CPMV HT, CPMV 160, CPMV 160+ and CPMV HT+.

A method (G) of increasing incorporation of VP4, VP7, or both VP4 andVP7 in a rotavirus like particle (RLP) is also described, the methodcomprising:

-   -   a) introducing into a host or host cell one or more nucleic acid        comprising    -   a first nucleotide sequence encoding a first rotavirus protein,        a second nucleotide sequence encoding a second rotavirus protein        and a third nucleotide sequence encoding a third rotavirus        protein, the first, second and third nucleotide sequence being        operatively linked to one or more regulatory region active in        the host or host cell; and    -   the first nucleotide sequence encoding rotavirus protein NSP4,        the second nucleotide sequence encoding rotavirus protein VP6,        and the third nucleotide sequence encoding one of rotavirus        protein VP7 or VP4;    -   b) incubating the host or host cell under conditions that permit        the expression of the one or more nucleic acid, so that each of        NSP4, VP6 and VP7 or VP4 are expressed, thereby producing the        RLP with enhanced levels of VP4, VP7, or both VP4 and VP7 when        compared to the level of VP4 or VP7 produced by a second host or        host cell that expresses the one or more nucleic acid that does        not comprise NSP4, under the same conditions.

In the method (G) as described above the one or more nucleic acid mayfurther comprises a fourth nucleotide sequence encoding a fourthrotavirus protein, the first, second, third, and fourth nucleotidesequence being operatively linked to one or more regulatory regionactive in the host or host cell; and

-   the first nucleotide sequence encoding rotavirus protein NSP4, the    second nucleotide sequence encoding rotavirus protein VP6, and the    third and fourth nucleotide sequence encoding rotavirus protein VP2,    VP4 or VP7 and wherein each of NSP4, VP6 and two of VP2, VP4 and VP7    are expressed from the one or more nucleic acid.

A method (H) of increasing incorporation of VP4, VP7, or both VP4 andVP7 in a rotavirus like particle (RLP) is also described, the methodcomprising:

-   -   a) introducing in a host or host cell comprising one or more        nucleic acid comprising

-   a first nucleotide sequence encoding a first rotavirus protein, a    second nucleotide sequence encoding a second rotavirus protein and a    third nucleotide sequence encoding a third rotavirus protein, the    first, second and third nucleotide sequence being operatively linked    to one or more regulatory region active in the host or host cell;    and

-   the first, second and third nucleotide sequence encoding one of    rotavirus protein NSP4, VP6 and one of rotavirus protein VP7 or VP4;    -   b) incubating the host or host cell under conditions that permit        the expression of the one or more nucleic acid, so that each of        NSP4, VP6 and VP7 or VP4 are expressed, thereby producing the        RLP with enhanced levels of VP4, VP7, or both VP4 and VP7 when        compared to the level of VP4 or VP7 produced by a second host or        host cell that expresses the one or more nucleic acid that does        not comprise NSP4, under the same conditions.

In the method (H) as described above the one or more nucleic acid mayfurther comprises a fourth nucleotide sequence encoding a fourthrotavirus protein, the first, second, third, and fourth nucleotidesequence being operatively linked to one or more regulatory regionactive in the host or host cell; and

-   the first, second, third and fourth nucleotide sequence encoding one    of rotavirus protein NSP4, VP6 and two of rotavirus protein VP2, VP7    or VP4 and wherein each of NSP4, VP6 and two of VP2, VP7 or VP4 are    expressed from the one or more nucleic acid.

In the method (G) and (H) as described above the one or more nucleicacid may further comprises a fourth nucleotide sequence encoding afourth rotavirus protein and a fifth nucleotide sequence encoding afifth rotavirus protein, the first, second, third, fourth and fifthnucleotide sequence being operatively linked to one or more regulatoryregion active in the host or host cell; and

-   the first, second, third, fourth and fifth nucleotide sequence    encoding one of rotavirus protein VP2, VP4, VP6, VP7 or NSP4 and    wherein each of VP2, VP4, VP6, VP7 and NSP4 are expressed from the    one or more nucleic acid.

In the method (G) and (H) as described above the one or more nucleicacid may further comprises a fourth nucleotide sequence encoding afourth rotavirus protein and a fifth nucleotide sequence encoding afifth rotavirus protein, the first, second, third, fourth and fifthnucleotide sequence being operatively linked to one or more regulatoryregion active in the host or host cell; and

-   the first, second, third, fourth and fifth nucleotide sequence    encoding one of rotavirus protein VP2, VP4, VP6, VP7 or NSP4 and    wherein each of VP2, VP4, VP6, VP7 and NSP4 are expressed from the    one or more nucleic acid.

In the method (G) or (H) as described above, in the step of introducing(step a), the one or more nucleic acid may comprise two nucleic acids,with a first nucleic acid comprising encoding the NSP4, and a secondnucleic acid encoding VP2, VP4, VP6 and VP7, the ratio of an amount ofthe first nucleic acid relative to the second nucleic acid that isintroduced into the plant, portion of a plant or plant cell is between1:0.8 and 1:2. The ratio may also be 1:1. Alternatively, the one or morenucleic acid may comprise two nucleic acids, with a first nucleic acidcomprising the first nucleotide sequence encoding the first rotavirusprotein and the second nucleotide sequence encoding the second rotavirusprotein, and a second nucleic acid comprising the third to fifthnucleotide sequence encoding the third to fifth rotavirus protein.

In the method (G) or (H) as described above, in the step of introducing(step a), the one or more nucleic acid may comprise three nucleic acids,with a first nucleic acid comprising the first and the second nucleotidesequence encoding the first and the second rotavirus protein, a secondnucleic acid comprising the third and fourth nucleotide sequenceencoding the third and the fourth rotavirus protein, and a third nucleicacid comprising the fifth nucleotide sequence encoding the fifthrotavirus protein, and wherein the ratio of an amount of the firstnucleic acid relative to the second nucleic acid and to the thirdnucleic that is introduced into the plant, the portion of the plant, orthe plant cell, is 1:1:1.

Alternatively, in the method (G) or (H) as described above, in the stepof introducing (step a), the one or more nucleic acid may comprise threenucleic acids, with the first nucleic acid comprising the firstnucleotide sequence encoding the first rotavirus protein, the secondnucleic acid comprising the second nucleotide sequence encoding thesecond rotavirus protein, and the third nucleic acid comprising thethird to fifth nucleotide sequence encoding the third to fifth rotavirusprotein, and wherein the ratio of an amount of the first nucleic acidrelative to the second nucleic acid and to the third nucleic that isintroduced into the plant, the portion of the plant, or the plant cell,is 1:1:1.

Furthermore, in the method (G) or (H) described above, in the step ofintroducing (step a), the one or more nucleic acid may comprise fournucleic acids, a first nucleic acid comprising the first nucleic acidencoding the first rotavirus protein, a second nucleic acid comprisingthe second nucleotide sequence encoding the second rotavirus protein, athird nucleic acid comprising the third nucleotide sequence encoding thethird rotavirus protein, and a fourth nucleic acid comprising the fourthand the fifth nucleotide sequence encoding the fourth and fifthrotavirus protein, wherein the ratio of an amount of the first nucleicacid relative to the second nucleic acid, to the third nucleic and tothe fourth nucleic acid that is introduced into the plant, the portionof the plant, or the plant cell, is 1:1:1:1.

In the method in the method (G) or (H) as described above, in the stepof introducing (step a), the one or more nucleic acid may comprise fivenucleic acids, a first nucleic acid comprising the first nucleotidesequence encoding the first rotavirus protein, a second nucleic acidcomprising the second nucleotide sequence encoding the second rotavirusprotein, a third nucleic acid comprising the third nucleotide sequenceencoding the third rotavirus protein, a fourth nucleic acid comprisingthe fourth nucleotide sequence encoding the fourth rotavirus protein,and a fifth nucleic acid comprising the fifth nucleotide sequenceencoding the fifth rotavirus protein, and the ratio of an amount of thefirst nucleic acid relative to the second nucleic acid, to the thirdnucleic acid, to the fourth nucleic acid and to the fifth nucleic acidthat is introduced into the plant, the portion of the plant, or theplant cell, is 1:1:1:1:1.

The one or more nucleotide sequence of the method (G) or (H) asdescribed above may be operatively linked to one or more expressionenhancer. Furthermore, the expression enhancer may be selected from thegroup consisting of CPMV HT, CPM 160, CPMV 160+ and CPMV HT+.

The methods (G) or (H) as described above may further comprise the stepsof:

-   c) harvesting the host or host cell, and-   d) purifying the RLPs from the host or host cell, wherein the RLPs    range in size from 70-100 nm.

In the method (A), the method (B), the method (C), the method (D), themethod (E), the method (F), the method (G) or the method (H) asdescribed above, the one or more nucleic acid may comprise one nucleicacid comprising the first, second, third, fourth and fifth nucleotidesequence encoding the first, second, third, fourth, and fifth rotavirusprotein.

In the method (A), the method (B), the method (C), the method (D), themethod (E), the method (F), the method (G) or the method (H) asdescribed above, the one or more nucleic acid may comprise two nucleicacids, for example, a first nucleic acid comprising the first nucleotidesequence encoding the first rotavirus protein, and a second nucleic acidcomprising the second to fifth nucleotide sequence encoding the secondto fifth rotavirus protein. Alternatively, the one or more nucleic acidmay comprise two nucleic acids, with a first nucleic acid comprising thefirst nucleotide sequence encoding the first rotavirus protein and thesecond nucleotide sequence encoding the second rotavirus protein, and asecond nucleic acid comprising the third to fifth nucleotide sequenceencoding the third to fifth rotavirus protein.

In the method (A), the method (B), the method (C), the method (D), themethod (E), the method (F), the method (G) or the method (H) asdescribed above, the one or more nucleic acid may also comprise threenucleic acids, a first nucleic acid comprising the first nucleotidesequence encoding the first rotavirus protein, the second nucleotidesequence encoding the second rotavirus protein, a second nucleic acidcomprising the third nucleotide sequence encoding the third rotavirusprotein and fourth nucleotide sequence encoding the fourth rotavirusprotein, and a third nucleic acid comprising the fifth nucleotidesequence encoding the fifth rotavirus protein. Alternatively, the one ormore nucleic acid may comprise three nucleic acids, with a first nucleicacid comprising the first nucleotide sequence encoding the firstrotavirus protein, a second nucleic acid comprising the secondnucleotide sequence encoding the second rotavirus protein, and a thirdnucleic acid comprising the third to fifth nucleotide sequence encodingthe third to fifth rotavirus protein.

In the method (A), the method (B), the method (C), the method (D), themethod (E), the method (F), the method (G) or the method (H) asdescribed above the one or more nucleic acid comprises four nucleicacids, a first nucleic acid comprising the first nucleotide sequenceencoding the first rotavirus protein, a second nucleic acid comprisingthe second nucleotide sequence encoding the second rotavirus protein, athird nucleic acid comprising the third nucleotide sequence encoding thethird rotavirus protein, and a fourth nucleic acid comprising the fourthand fifth nucleotide sequence encoding the fourth and fifth rotavirusprotein.

Furthermore, in the method (A), the method (B), the method (C), themethod (D), the method (E), the method (F), the method (G) or the method(H) as described above the one or more nucleic acid may comprise fivenucleic acids, with a first nucleic acid comprising the first nucleotidesequence encoding the first rotavirus protein, a second nucleic acidcomprising the second nucleotide sequence encoding the second rotavirusprotein, a third nucleic acid comprising the third nucleotide sequenceencoding the third rotavirus protein, a fourth nucleic acid comprisingthe fourth nucleotide sequence encoding the fourth rotavirus protein,and a fifth nucleic acid comprising the fifth nucleotide sequenceencoding the fifth rotavirus protein.

The one or more nucleotide sequence of the method (A), the method (B),the method (C), the method (D), the method (E), the method (F), themethod (G) or the method (H) as described above may be operativelylinked to one or more expression enhancer. Furthermore, the expressionenhancer may be selected from the group consisting of CPMV HT, CPM 160,CPMV 160+ and CPMV HT+.

In the method (A), the method (B), the method (C), the method (D), themethod (E), the method (F), the method (G) or the method (H) asdescribed above the host or host cell may comprise insect cells,mammalian cells, plant, portion of a plant or plant cells. The plant maybe Nicotiana benthamiana.

Also described herein is an RLP produced by the method (A), the method(B), the method (C), the method (D), the method (E), the method (F), themethod (G) or the method (H) described above, wherein the RLP is atriple layered RLP comprising rotavirus protein, the rotavirus proteinconsists of VP2, VP4, VP6 and VP7. The RLP may not comprise NSP4.

A composition comprising an effective dose of the RLP for inducing animmune response in a subject, and a pharmaceutically acceptable carrier,and a method of inducing immunity to a rotavirus infection in a subject,that comprises administering the composition, are also described. In themethod of inducing immunity, the composition may be administered to asubject orally, intradermally, intranasally, intramuscularly,intraperitoneally, intravenously, or subcutaneously.

Also described herein is plant matter comprising an RLP produced by themethod (A), the method (B), the method (C), the method (D), the method(E), the method (F), the method (G) or the method (H) as describedabove.

In the method (A), the method (B), the method (C), the method (D), themethod (E), the method (F), the method (G) or the method (H) asdescribed above, the one or more nucleic acid may comprise one nucleicacid comprising the first, second and third nucleotide sequence encodingthe first, second and third, rotavirus protein. Furthermore, in themethod (A), the method (B), the method (C), the method (D), the method(E), the method (F), the method (G) or the method (H) as described abovethe one or more nucleic acid may comprise two nucleic acids, a firstnucleic acid comprising the first nucleotide sequence encoding the firstrotavirus protein, and a second nucleic acid comprising the second andthird nucleotide sequence encoding the second and third rotavirusprotein. Furthermore, in the method (A), the method (B), the method (C),the method (D), the method (E), the method (F), the method (G) or themethod (H) as described above the one or more nucleic acid may comprisetwo nucleic acids, a first nucleic acid comprising the first and secondnucleotide sequence encoding the first and second rotavirus protein, anda second nucleic acid comprising the third nucleotide sequence encodingthe third rotavirus protein. Alternatively, in the method (A), themethod (B), the method (C), the method (D), the method (E), the method(F), the method (G) or the method (H) as described above the one or morenucleic acid may comprise three nucleic acids, a first nucleic acidcomprising the first nucleotide sequence encoding the first rotavirusprotein, a second nucleic acid comprising the second nucleotide sequenceencoding the second rotavirus protein and a third nucleic acidcomprising the third nucleotide sequence encoding the third rotavirusprotein.

As described herein, by co-expressing NSP4 along with VP6 and VP4 orVP7, in a host or host cell, for example a plant, portion of the plant,or a plant cell, RLPs comprising increased levels of VP4, VP7, or bothVP4 and VP7 are observed, when compared to the level of VP4 and VP7 inRLPs produced by a second host or host cell for example a plant, portionof a second plant, or second plant cell, that expresses the one or morenucleic acid that encodes VP6 and VP4 or VP7, and does not encode NSP4,the second host or second host cell for example a second plant, thesecond portion of plant, or the second plant cell, incubated or grown,under the same conditions as the host or host cell for example a plant,portion of the plant, or plant cell.

This summary of the invention does not necessarily describe all featuresof the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent fromthe following description in which reference is made to the appendeddrawings wherein:

FIG. 1 shows rotavirus cell entry and replication. When rotavirus entersa cell, VP4 and VP7 are lost, forming a double layered particle (DLP).Transcription of the dsRNA commences resulting in translation of VP2,VP4, VP6 and VP7. Progeny cores with replicase activity are produced invirus factories (also called viroplasms). Late transcription occurs inthese progeny cores. At the periphery of virus factories, these core arecoated with VP6, forming immature DLPs that bud across the membrane ofthe endoplasmic reticulum, acquiring a transient lipid membrane which ismodified with the ER resident viral glycoproteins NSP4 and VP7; theseenveloped particles also contain VP4. As the particles move towards theinterior of the ER cisternae, the transient lipid membrane and thenonstructural protein NSP4 are lost, while the virus surface proteinsVP4 and VP7 rearrange to form the outermost virus protein layer,yielding mature infectious triple-layered particles (see Swiss Instituteof Bioinformatics (ViralZone):viralzone.expasy.org/viralzone/all_by_species/107.html)

FIG. 2 shows rotavirus-like particle purification by ultracentrifugationon iodixanol density gradient. FIG. 2A presents the percentage ofiodixanol and volume for each layer of the gradient used for thepurification of rotavirus-like particles. After centrifugation, thegradient was fractionated into 1 ml fractions starting from the bottomof the tube. The approximate localization of fractions 1 to 13 areindicated by arrows. FIG. 2B shown a Coomassie-stained SDS-PAGE analysisof the protein content of fractions 1 to 10 from an iodixanol densitygradient separation applied to a crude protein extract from leavesexpressing rotavirus VP2, VP4, VP6 and VP7 antigens.

FIG. 3 shows rotavirus protein expression. FIG. 3A shows aCoomassie-stained SDS-PAGE analysis of fractions 2 and 3 from aniodixanol density gradient separation applied to crude protein extractsfrom leaves expressing VP2, VP4, VP6, VP7 in the presence or absence ofNSP4. Rotavirus structural proteins VP2, VP6, VP7, VP4 were expressed,left panel, using individual constructs for each structural antigen(“single gene constructs”), with NSP4 on a separate construct; middlepanel, two constructs, each having the genes of two structural antigens(“dual gene constructs”), with NSP4 on a separate construct; or rightpanel, a single construct for the co-expression of the four structuralantigens (Quadruple gene constructs), with a separate construct for theexpression of NSP4. Position of the rotavirus VP2 and VP6 antigen areshown by arrows. FIG. 3B shows a Western blot analysis of fraction F2from the same treatments as in FIG. 3A using an anti-rotavirus VP4 orVP7 antibody as specified.

FIG. 4 shows rotavirus protein expression in the presence of anexpression enhancer. FIG. 4A shows a Coomassie-stained SDS-PAGE analysisof fractions F2 and F3 from an iodixanol density gradient applied tocrude protein extracts from leaves co-expressing VP2, VP4, VP6, VP7 andNSP4. Rotavirus structural proteins VP2, VP6, VP7, VP4 were expressedfrom single gene constructs, and one construct expressing NSP4 (leftpanel), or from two dual gene constructs and one construct expressingNSP4 (middle and right panel)). Each construct comprised an expressionenhancer, either CPMV HT (left and middle panels) or CMPV 160 (rightpanel), except for NSP4 which always comprised the CPMV-HT enhancer. Theratios indicate the proportion of the Agrobacterium strains in thebacterial suspension used for transformation: left panel—five singlegene constructs (VP2, VP6, VP4, VP7 and NSP4; ratio of 1:1:1:1:1);middle and right panels—dual gene constructs encoding structuralproteins (VP6/2 and VP7/4) and the construct encoding non-structuralprotein (NSP4; ratio of 1:1:1). FIG. 4B shows a Western blot analysis ofF2 from the same treatments as in FIG. 4A using an anti-rotavirus VP4 orVP7 antibody as specified. The ratios indicate the proportion of theAgrobacterium strains in the bacterial suspension used fortransformation: left panel—five single gene constructs (VP2, VP6, VP4,VP7 and NSP4; ratio of 1:1:1:1:1); middle and right panels—dual geneconstructs encoding structural proteins (VP6/2 and VP7/4) and theconstruct encoding non-structural protein (NSP4; ratio of 1:1:1).

FIG. 5 shows rotavirus protein expression. Upper panel shows aCoomassie-stained SDS-PAGE analysis of fractions F2 and F3 an iodixanoldensity gradient applied to crude protein extracts from leavesco-expressing VP2, VP4, VP6, VP7 and NSP4, and the lower panel shows aWestern blot analysis of the corresponding F2 fraction from the upperpanel. Rotavirus structural proteins VP2, VP6, VP7 and VP4 wereexpressed within a quadruple gene construct, and the non-structuralprotein NSP4 was co-expressed from a distinct single gene construct(lanes 1 and 2), or structural proteins VP2, VP6, VP7, VP4 and thenon-structural protein NSP4 were expressed within a quintuple geneconstruct (lane 3). The ratio of agroinfiltration of the constructs isindicated. An OD of 0.4 of Agrobacterium strains in the bacterialsuspension is indicated as 1. An OD of 0.6 of Agrobacterium strains inthe bacterial suspension is indicated as 1.5.

FIG. 6 shows a general schematic of an example of several enhancersequences that may be used in the constructs of the present invention.FIG. 6A and FIG. 6B show a general schematic of the CPMV HT and CPMV HT+enhancer sequences fused to a nucleotide sequence of interest (forexample encoding a rotavirus structural protein VP2, VP4, VP6, VP7, or anon-structural protein NSP4). Not all of the elements shown in FIG. 5Aor 5B may be required within the enhancer sequence. Additional elementsmay be included at the 3′ end of the nucleotide sequence of interestincluding a sequence encoding a comovirus 3′ untranslated region (CPMV3′ UTR), or a plastocyanin 3′ UTR (3′UTR). FIGS. 6C and 6D show ageneral schematic of the enhancer sequence of CPMVX, and CPMVX+(comprising CPMVX, and a stuffer fragment, which in this non-limitingexample, comprises a multiple cloning site and a plant kozak sequence),as described herein. CPMVX and CPMVX+ are each shown as operativelylinked to plant regulatory region at their 5′ ends, and at their 3′ends, in series, a nucleotide sequence of interest (including an ATGinitiation site and STOP site), a 3′UTR, and a terminator sequence. Anexample of construct CPMVX as described herein, is CPMV160. An exampleof construct CPMVX+ as described herein, is CPMV160+.

FIG. 7 shows sequence components used to prepare construct number 1710(2X35S/CPMV-HT/RVA(WA) VP2(opt)/NOS. FIG. 7A shows the nucleotidesequence of IF-WA_VP2(opt).s1+3c (SEQ ID NO: 19). FIG. 7B shows thenucleotide sequence of IF-WA_VP2(opt).s1−4r (SEQ ID NO: 20). FIG. 7Cshows the optimized coding sequence of Rotavirus A VP2 from strain WA(SEQ ID NO: 21). FIG. 7D shows the schematic representation of construct1191. FIG. 7E shows the nucleotide sequence of construct 1191 (SEQ IDNO: 22). FIG. 7F shows the nucleotide sequence of expression cassettenumber 1710 (SEQ ID NO: 23). FIG. 7G shows the amino acid sequence ofVP2 from Rotavirus A WA strain (SEQ ID NO: 24). FIG. 7H shows theschematic representation of construct number 1710.

FIG. 8 shows sequence components used to prepare construct number 1713(2X35S/CPMV-HT/RVA(WA) VP6(opt)/NOS). FIG. 8A shows the nucleotidesequence of IF-WA_VP6(opt).s1+3c (SEQ ID NO: 25). FIG. 8B shows thenucleotide sequence of IF-WA_VP6(opt).s1−4r (SEQ ID NO: 26). FIG. 8Cshows the optimized coding sequence of Rotavirus A VP6 from strain WA(SEQ ID NO: 217. FIG. 8D shows the nucleotide sequence of expressioncassette number 1713 (SEQ ID NO: 28). FIG. 8E shows the amino acidsequence of VP6 from Rotavirus A WA strain (SEQ ID NO: 29). FIG. 8Fshows the schematic representation of construct number 1713.

FIG. 9 shows sequence components used to prepare construct number 1730(2X35S/CPMV-HT/RVA(Rtx) VP4(opt)/NOS). FIG. 9A shows the nucleotidesequence of IF-Rtx_VP4(opt).s1+3c (SEQ ID NO: 30). FIG. 9B shows thenucleotide sequence of IF-Rtx_VP4(opt).s1−4r (SEQ ID NO: 31). FIG. 9Cshows the optimized coding sequence of Rotavirus A VP4 from strainRVA/Vaccine/USA/Rotarix-A41CB052A/1988/G1P1A[8] (SEQ ID NO:32). FIG. 9Dshows the nucleotide sequence of expression cassette number 1730 (SEQ IDNO: 33). FIG. 9E shows the amino acid sequence of VP4 from Rotavirus ARotarix strain (SEQ ID NO: 34). FIG. 9F shows the schematicrepresentation of construct number 1730.

FIG. 10 shows sequence components used to prepare construct number 1734(2X35S/CPMV-HT/RVA(Rtx) VP7(Opt)/NOS). FIG. 10A shows the nucleotidesequence of IF-TrSP+Rtx_VP7(opt).s1+3c (SEQ ID NO: 35). FIG. 10B showsthe nucleotide sequence of IF-Rtx_VP7(opt).s1−4r (SEQ ID NO: 36). FIG.10C shows the optimized coding sequence of Rotavirus A VP7 from strainRVA/Vaccine/USA/Rotarix-A41CB052A/1988/G1P1A[8] (SEQ ID NO: 37). FIG.10D shows the nucleotide sequence of expression cassette number 1734(SEQ ID NO: 38). FIG. 10E shows the amino acid sequence of TrSp-VP7 fromRotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] strain (SEQ IDNO: 39). FIG. 10F shows the schematic representation of construct number1734.

FIG. 11 shows sequence components used to prepare construct number 1706(2X35S/CPMV-HT/RVA(WA) NSP4/NOS). FIG. 11A shows the nucleotide sequenceof IF-WA_NSP4.s1+3c (SEQ ID NO: 40). FIG. 11B shows the nucleotidesequence of IF-WA_NSP4.s1−4r (SEQ ID NO: 41). FIG. 11C shows the codingsequence of Rotavirus A VP6 from strain WA (SEQ ID NO: 42). FIG. 11Dshows the nucleotide sequence of expression cassette number 1706 (SEQ IDNO: 43). FIG. 11E shows the amino acid sequence of NSP4 from Rotavirus AWA strain (SEQ ID NO: 44). FIG. 11F shows the schematic representationof construct number 1706.

FIG. 12 shows sequence components used to prepare construct number 1108(2X35S/CPMV-160/RVA(WA) VP2(opt)/NOS). FIG. 12A shows the nucleotidesequence of IF(C160)-WA_VP2(opt).c (SEQ ID NO: 45). FIG. 12B shows aschematic representation of construct 1190. FIG. 12C shows thenucleotide sequence of construct 1190 (SEQ ID NO: 46). FIG. 12D showsthe nucleotide sequence of expression cassette number 1108 (SEQ ID NO:47). FIG. 12E shows a schematic representation of construct number 1108.

FIG. 13 shows sequence components used to prepare construct number 1128(2X35S/CPMV-160/RVA(WA) VP6(opt)/NOS). FIG. 13A shows the nucleotidesequence of IF(C160)-WA_VP6(opt).c (SEQ ID NO: 48). FIG. 13B shows thenucleotide sequence of expression cassette number 1128 (SEQ ID NO: 49).FIG. 13C shows a schematic representation of construct number 1128.

FIG. 14 shows sequence components used to prepare construct number 1178(2X35S/CPMV-160/RVA(Rtx) VP4(opt)/NOS). FIG. 14A shows the nucleotidesequence of IF(C160)-Rtx_VP4(opt).c (SEQ ID NO: 50). FIG. 14B shows thenucleotide sequence of expression cassette number 1178 (SEQ ID NO: 51).FIG. 14C shows the schematic representation of construct number 1178.

FIG. 15 shows sequence components used to prepare construct number 1199(2X35S/CPMV-160/TrSp-RVA(Rtx) VP7(Opt)/NOS). FIG. 15A shows thenucleotide sequence of IF(C160)-TrSP+Rtx_VP7(opt).c (SEQ ID NO: 52).FIG. 15B shows the nucleotide sequence of Expression cassette number1199 (SEQ ID NO: 53). FIG. 15C shows the schematic representation ofconstruct number 1199.

FIG. 16 shows the schematic representation of construct number 1708(double gene construct for the expression of VP6 and VP2 under CPMV-HTexpression cassette).

FIG. 17 shows the schematic representation of construct number 1719(double gene construct for the expression of VP7 and VP4 under CPMV-HTexpression cassette).

FIG. 18 shows the schematic representation of construct number 2400(double gene construct for the expression of VP6 and VP2 under CPMV-160expression cassette).

FIG. 19 shows the schematic representation of construct number 2408(double gene construct for the expression of VP7 and VP4 under CPMV-160expression cassette).

FIG. 20 shows the schematic representation of construct number 1769(quadruple gene construct for the expression of VP7, VP4, VP6 and VP2under CPMV-HT expression cassette).

FIG. 21 shows the schematic representation of construct number 2441(quintuple gene construct for the expression of VP4, VP7, NSP4, VP6 andVP2 under CPMV-HT expression cassette).

DETAILED DESCRIPTION

The following description is of a preferred embodiment.

The present invention relates to virus-like particles (VLPs) comprisingone or more rotavirus structural protein (i.e. a rotavirus likeparticle, rotavirus VLP or RLP), and methods of producing rotavirus-likeparticle (RLPs) in any host, particularly in plants, a portion of aplant, or a plant cell. Other hosts might comprise, for example, insectcells and mammalian cells. The rotavirus like particle (RLP) maycomprise one or more rotavirus structural protein. The RLP may triplelayered. The RLP may be produced by co-expressing rotavirus structuraland nonstructural proteins in plant, however, the RLP does not compriseany rotavirus nonstructural proteins.

The host or host cell may be from any source including plants, fungi,bacteria, insect and animals. In a preferred embodiment the host or hostcell is a plant or plant cell.

The present invention in part provides further a method of producing arotavirus-like particle (RLP) in a host, such as a plant, a portion of aplant, or a plant cell. The method may comprise introducing one or morenucleic acid comprising a regulatory region active in the host, such asa plant, a portion of a plant, or a plant cell, the regulatory regionoperatively linked to a nucleotide sequence encoding one or morerotavirus structural protein and one or more rotavirus nonstructuralprotein into the host, such as into a plant, portion of the plant, orplant cell. Followed by incubating the host, such as a plant, portion ofthe plant, or plant cell under conditions that permit the expression ofthe nucleic acids, thereby producing the RLP comprising one or morerotavirus structural protein. The one or more rotavirus structuralprotein may be rotavirus protein VP2, VP4, VP6 or VP7. The rotavirusnonstructural protein may be NSP4. The RLP may be triple layered. TheRLP may comprise rotavirus structural protein VP2, VP4, VP6 and VP7, anddoes not comprise the nonstructural protein NSP4.

The present invention in part provides further a method of producing arotavirus like particle (RLP) in a host or host cell, the method maycomprise:

-   providing a host or host cell comprising one or more nucleic acid    comprising a first nucleotide sequence encoding a first rotavirus    protein, a second nucleotide sequence encoding a second rotavirus    protein and a third nucleotide sequence encoding a third rotavirus    protein, the first, second and third nucleotide sequence being    operatively linked to one or more regulatory region active in the    host or host cell; and-   the first, second and third nucleotide sequence encoding NSP4 and    one or two of rotavirus protein VP2 or VP6 and one or two of    rotavirus protein VP7 or VP4;-   incubating the host or host cell under conditions that permit the    expression of the one or more nucleic acid, so that NSP4 and either    VP2, VP4 and VP7, or VP2, VP6 and VP7, or VP2, VP6 and VP4, or VP6,    VP4 and VP7, are expressed, thereby producing the RLP.

Furthermore, the one or more nucleic acid may comprise a fourthnucleotide sequence encoding a fourth rotavirus protein. The first,second, third, and fourth nucleotide sequence being operatively linkedto one or more regulatory region active in the host or host cell; andthe first nucleotide sequence, the second nucleotide sequence, the thirdand fourth nucleotide sequence encoding rotavirus protein VP2, or VP6,VP4 or VP7 and NSP4, wherein NSP4 and wherein either VP2 or VP6 and VP4or VP7 are expressed from the one or more nucleic acid.

Furthermore, the present invention in part provides a method ofproducing a rotavirus-like particle (RLP) vaccine candidate in a host,such as a plant, a portion of the plant, or a plant cell. The method maycomprise expressing in a host, such as in a plant or portion of a plant,one or more nucleic acid (R₁-R₅) comprising one or more regulatoryregion active in the host, such as in the plant, portion of a plant, orplant cell, the regulatory region operatively linked to nucleotidesequences R₁-R₅, wherein nucleotide sequence R₁ encodes rotavirusprotein X₁, nucleotide sequence R₂ encodes rotavirus protein X₂,nucleotide sequence R₃ encodes rotavirus protein X₃, nucleotide sequenceR₄ encodes rotavirus protein X₄ and nucleotide sequence R₅ encodesrotavirus protein X₅ and each of X₁-X₅ are selected from the group ofrotavirus protein VP2, VP4, VP6, VP7 and NSP4, so that each of VP2, VP4,VP6, VP7 and NSP4 are expressed in the host, such as in the plant,portion of the plant, or plant cell (see Table 1). The RLP may compriserotavirus structural protein VP2, VP4, VP6 and VP7. The RLP does notcomprise nonstructural protein NSP4.

It has been found that by introducing and co-expressing rotavirusstructural protein and a rotavirus non-structural protein in the host,such as a plant or portion of the plant that the yield of the RLPproduced may be modulated. In particular, it has been found that byco-expressing rotavirus structural proteins along with a rotavirusnon-structural protein NSP4 in the host, such as a plant, portion of theplant, or plant cell, that the incorporation of structural protein VP4,VP7 or both VP4 and VP7 into the RLP may be increased, when compared tothe level of VP4 and VP7 produced by a second host, such as a secondplant, portion of a second plant, or second plant cell that expressesthe same rotavirus structural proteins but that does not express therotavirus non-structural protein, under the same conditions.

For example a method of increasing incorporation of VP4, VP7, or bothVP4 and VP7 in a rotavirus like particle (RLP) is provided. The methodcomprises:

-   -   a) providing a host or host cell comprising one or more nucleic        acid comprising    -   a first nucleotide sequence encoding a first rotavirus protein,        a second nucleotide sequence encoding a second rotavirus        protein, a third nucleotide sequence encoding a third rotavirus        protein and a fourth nucleotide sequence encoding a fourth        rotavirus protein; the first, second, third and fourth        nucleotide sequence being operatively linked to one or more        regulatory region active in the host or host cell; and    -   the first, second, third and fourth nucleotide sequence encoding        one of rotavirus protein VP7, VP4, NSP4 and VP2 or VP6;    -   b) incubating the host or host cell under conditions that permit        the expression of the one or more nucleic acid, so that each of        the VP7, VP4, NSP4 and VP2 or VP6 and are expressed, thereby        producing the RLP with enhanced levels of VP4, VP7, or both VP4        and VP7 when compared to the level of VP4 or VP7 produced by a        second host or host cell that expresses the one or more nucleic        acid that does not comprise NSP4, under the same conditions.

Furthermore, an alternative method of increasing production of VP4, VP7or both VP4 and VP7 in a rotavirus like particle (RLP) may comprises:

-   -   a) introducing into a plant, portion of a plant or plant cell        one or more nucleic acid comprising one or more regulatory        operatively linked to a first, second, third, fourth and fifth        nucleotide sequence, the one or more regulatory region active in        the plant, portion of the plant or the plant cell, the first        nucleotide sequence encoding a first rotavirus protein, the        second nucleotide sequence encoding a second rotavirus protein,        the third nucleotide sequence encoding a third rotavirus        protein, the fourth nucleotide sequence encoding a fourth        rotavirus protein and the fifth nucleotide sequence encoding a        fifth rotavirus protein, each of the first, second, third,        fourth or fifth nucleotide sequence encoding one of VP2, VP4,        VP6, VP7 or NSP4, and    -   b) incubating the plant, portion of a plant or plant cell under        conditions that permit the transient expression of the one or        more nucleic acid so that each of VP2, VP4, VP6, VP7 and NSP4        are transiently expressed, thereby producing the RLP with        enhanced levels of VP4 and VP7 when compared to the level of VP4        and VP7 produced by a second plant, portion of a second plant,        or second plant cell that expresses the one or more nucleic acid        that does not comprise NSP4, under the same conditions.

If desired, the method may further comprises the steps of:

-   -   c) harvesting the plant, portion of a plant or plant cell, and    -   d) purifying the RLPs from the plant, portion of a plant or        plant cell, wherein the RLPs range in size from 70-100 nm.

An alternate method of increasing production of VP4, VP7 or both VP4 andVP7 in a rotavirus like particle (RLP) is also provided, the methodcomprising:

-   -   a) providing a plant, portion of a plant or plant cell        comprising one or more nucleic acid comprising one or more        regulatory region operatively linked to a first, second, third,        fourth and fifth nucleotide sequence, the one or more regulatory        region active in the plant, portion of the plant or the plant        cell, the first nucleotide sequence encoding a first rotavirus        protein, the second nucleotide sequence encoding a second        rotavirus protein, the third nucleotide sequence encoding a        third rotavirus protein, the fourth nucleotide sequence encoding        a fourth rotavirus protein and the fifth nucleotide sequence        encoding a fifth rotavirus protein, each of the first, second,        third, fourth and fifth nucleotide sequence encoding one of VP2,        VP4, VP6, VP7 or NSP4, and    -   b) incubating the plant, portion of a plant or plant cell under        conditions that permit the expression of the one or more nucleic        acid, so that each of the VP2, VP4, VP6, VP7 and NSP4 are        expressed, thereby producing the RLP with enhanced levels of VP4        and VP7 when compared to the level of VP4 and VP7 produced by a        second plant, portion of a second plant, or second plant cell        that expresses the one or more nucleic acid that does not        comprise NSP4, under the same conditions.

It has been further found that by modulating the ratio betweenconstructs comprising nucleic acids encoding rotavirus structuralproteins and the non-structural protein NSP4 during transient expressionin the host, such as a plant, portion of the plant, or plant cell, theyield of RLP production and the incorporation of structural proteins VP4and VP7 into the RLP may be improved.

Without wishing to be bound by theory, the co-expression of a rotavirusnon-structural protein for example NSP4 together with one or morerotavirus structural protein for example VP2, VP4, VP6 and/or VP7 maylead to an increase in incorporation of VP4 and/or VP7 into RLPs. Thisincrease of incorporation of VP4 and/or VP7 into RLPs may occur via anincrease of expression or production of the rotavirus proteins and/orbecause of an increase of the efficacy of the assembly of RLPs and/or anincrease of recruiting of the rotavirus proteins at the RLP assemblysite.

As shown in FIGS. 3A and 3B, the co-expression of rotavirusnon-structural protein NSP4 together with rotavirus structural proteinVP2, VP6, VP4 and VP7 in plants lead to an increase in incorporation ofVP4 and VP7 into RLPs, when compared to the expression of structuralprotein VP2, VP6, VP4 and VP7 without the presence of NSP4 (see FIG.3B).

The ratios of the constructs encoding the various structural andnon-structural proteins that are transiently expressed in the host, suchas a plant, portion of the plant, or plant cell, may be altered byproviding constructs comprising nucleic acid sequences encodingrotavirus structural proteins and the non-structural protein NSP4 ontwo, three, four or five constructs and varying the amount of eachconstruct during the step of introducing the construct in the host(using Agrobacterium to the plant, plant portion or plant cell). Forexample, five separate constructs, each encoding one structural proteinand the non-structural protein, may be co-introduced at various ratiosresulting co-expression of the nucleic acids at various ratios within aplant, plant portion, or plant cell. Alternatively, the nucleic acidsequences may be provided in various combinations on two, three or fourconstructs and the constructs co-introduced in the plant, portion of theplant, or plant cell, at various ratios, as described below.

Additionally, the nucleic acid sequences may be provided on the sameconstruct.

“Rotavirus protein” may refer to rotavirus structural protein orrotavirus nonstructural proteins. A “rotavirus structural protein” mayrefer to all or a portion of a rotavirus structural protein isolatedfrom rotavirus, present in any naturally occurring, or a variant of anynaturally occurring, rotavirus strain or isolate. Thus, the termrotavirus structural protein includes a naturally occurring rotavirusstructural protein, or a variant of a rotavirus structural protein thatmay be produced by mutation during the virus life-cycle or produced inresponse to selective pressure (e.g., drug therapy, expansion of hostcell tropism, or infectivity, etc.). As one of skill in the artappreciates, such rotavirus structural proteins and variants thereof maybe also produced using recombinant techniques. Rotavirus structuralproteins may include capsid proteins such for example VP2 and VP6,surface proteins, for example VP4, or a combination of capsid andsurface proteins. The structural protein may further include for exampleVP7.

By rotavirus “non structural protein”, “nonstructural protein”,“non-structural protein”, “NSP” or “nonstructural rotavirus protein” itis meant a protein that is encoded by the rotavirus genome, but notpackaged into the viral particle. Non-limiting examples of rotavirusnonstructural proteins are rotavirus NSP4.

By “co-expressed” it is meant that two, or more than two, nucleotidesequences are expressed at about the same time within the plant, withinthe same tissue of the plant and within the same cells in the plant. Thenucleotide sequences need not be expressed at exactly the same time.Rather, the two or more nucleotide sequences are expressed in a mannersuch that the encoded products have a chance to interact within adesired cellular compartment. For example, the non-structural proteinmay be preferably expressed either before or during the period when thestructural proteins are expressed. The two or more than two nucleotidesequences can be co-expressed using a transient expression system, wherethe two or more sequences are introduced within the plant at about thesame time, under conditions that the two or more sequences areexpressed. The two or more than two sequences may be present ondifferent constructs, and co-expression requires introduction of each ofthe constructs into the plant, portion of plant or plant cell, or thetwo or more than two sequences may be present on one construct and theconstruct introduced into the plant, portion of plant or plant cell.

The term “virus-like particle” (VLP), or “virus-like particles” or“VLPs” refers to structures that self-assemble and comprise one or morestructural proteins such as for example rotavirus structural protein,for example but not limited to VP2, VP4, VP6, VP7, or a combination ofVP2, VP4, VP6, VP7, structural protein. VLPs comprising rotavirusstructural protein maybe also be referred to “rotavirus VLP”,“rotavirus-like particle (RVLP)”, “rotavirus-like particle (RLP)”,“rotavirus-like particle”, “RVLP” or “RLP”. VLPs or RLPs are generallymorphologically and antigenically similar to virions produced in aninfection, but lack genetic information sufficient to replicate and thusare non-infectious. VLPs may be produced in suitable eukaryotic hostcells including plant host cells. Following extraction from the hostcell and upon isolation and further purification under suitableconditions, VLPs may be recovered as intact structures. The RLP may be asingle, double, or triple-layered RLP. Triple-layered RLPs may beobtained by the simultaneous expression of three or more rotavirusstructural proteins, and as described herein, co-expression with one ormore non-structural protein. For example, the co-expression ofstructural proteins VP2, VP6, VP7, VP4 and nonstructural protein NSP4results in producing triple-layered RLPs.

Co-expression of VP4, along with VP2, VP6, VP7, and one or morenon-structural protein as required, results in a particle with spikesthat resembles native rotavirus. VP4 may be processed or cleaved toproduce VP5 and VP8. This processing may take place within the hostusing endogenous proteases, or by co-expressing a suitable protease, forexample, trypsin, a trypsin-like protease, a serine protease, achymotrypsin-like protease, subtilisin. Alternatively, VP4 may beprocessed to produce VP5 and VP8 by adding a suitable protease, forexample, trypsin, a trypsin-like protease, a serine protease, achymotrypsin-like protease, subtilisin during any step of the RLPextraction procedure, or after RLP purification.

Each of the rotavirus structural proteins has different characteristicsand size, and is required in different amounts for assembly into RLP.The term “rotavirus VLP”, “rotavirus virus-like particle (RVLP)”,“rotavirus virus-like particle (RLP)”, “rotavirus virus-like particle”,“RVLP” or “RLP” refers to a virus-like particle (VLP) comprising one ormore rotavirus structural proteins. Example of rotavirus structuralproteins may include, but are not limited to VP2, VP4 (or VP5 and VP8)VP6 and VP7 structural protein. The RLP may not comprise rotavirusnonstructural proteins.

The present invention provides for a method of producing RLPs in aplant, wherein one or more nucleic acid (N₁-N₅) comprising one or moreregulatory region active in the plant are operatively linked tonucleotide sequences R₁-R₅, wherein nucleotide sequence R₁ encodesrotavirus protein X₁, nucleotide sequence R₂ encodes rotavirus proteinX₂, nucleotide sequence R₃ encodes rotavirus protein X₃, nucleotidesequence R₄ encodes rotavirus protein X₄ and nucleotide sequence R₅encodes rotavirus protein X₅ and wherein X₁-X₅ are selected from thegroup of rotavirus protein VP2, VP4, VP6, VP7 and NSP4, wherein VP2,VP4, VP6, VP7 and NSP4 are each selected once (see Table 1). The finalset, or combination, of nucleic acids used to transform the host resultsin the expression of each rotavirus protein within the host resulting inexpression of VP2, VP4, VP6, VP7 and NSP4 and formation of an RLP.

For example, with reference to Table 1, 2 nucleic acids (N₁ and N₂), maybe used to transform a host, (see example #2.1). In this case N₁comprises the R₁ nucleotide sequence and R₁ may encode one of VP2, VP4,VP6, VP7, or NSP4. The nucleic acid N₂ comprises four sequences R₂ toR₅, each of R₂ to R₅ encoding one of VP2, VP4, VP6, VP7, or NSP4, butnot the protein encoded by R₁, so that each of the VP2, VP4, VP6, VP7and NSP4 are expressed within the host, thereby producing the RLP. As anon-limiting example, N₁ may comprise R₁ which may encode VP2, and N₂may comprise R₂ to R₅ which may encode VP4, VP6, VP7 and NSP4respectively.

Table 1, provides an overview of combinations, which is not to beconsidered limiting, of nucleic acids (N), and nucleotide sequences (R)that may be expressed or co-expressed within a host to produce an RLPcomprising VP2, VP4, VP6, and VP7.

TABLE 1 Total # Nucleic Combination # Acids 1.1 Nucleic N₁ 1 1.2 AcidsN₁ 1 1.3 N₁ 1 2.1 N₁ N₂ 2 2.2 N₁ N₂ 2 2.3 N₁ N₂ 2 2.4 N₁ N₂ 2 2.5 N₁ N₂2 3.1 N₁ N₂ N₃ 3 3.2 N₁ N₂ N₃ 3 3.3 N₁ N₂ N₃ 3 3.4 N₁ N₂ N₃ 3 4.1 N₁ N₂N₃ N₄ 4 4.2 N₁ N₂ N₃ N₄ 4 5 N₁ N₂ N₃ N₄ N₅ 5 Nucleotide Sequence R₁ R₂R₃ R₄ R₅ Rotavirus Protein Protein X₁ X₂ X₃ X₄ X₅ type X₍₁₋₅₎ may VP2VP2 VP2 VP2 VP2 Structural be* VP4 VP4 VP4 VP4 VP4 Structural VP6 VP6VP6 VP6 VP6 Structural VP7 VP7 VP7 VP7 VP7 Structural NSP4 NSP4 NSP4NSP4 NSP4 Non- structural *For combinations 1.1, 2.1, 2.2, 3.1, 3.2, 4.1and 5: X₁, X₂, X₃, X₄ and X₅ each have to be a different rotavirusprotein selected from the group of VP2, VP4, VP6, VP7 and NSP4. Forcombination 1.3, 2.5 and 3.4: X₁, X₂ and X₃ each have to be a differentrotavirus protein selected from either VP4, VP6 and NSP4 or VP7, VP6 andNSP4. For combination 1.2, 2.3, 2.4, 3.3 and 4.2: X₁, X₂, X₃ and X₄ eachhave to be a different rotavirus protein selected from the group of VP2,VP4, VP6 and NSP4, VP2, VP7, VP6 and NSP4 or VP4, VP7, VP6 and NSP4.

1. One Construct

1.1 Quintuple Gene Construct

As described herein, a method of producing RLPs in a plant is provided,wherein a nucleic acid (N₁; a first nucleic acid) comprising a first,second, third, fourth and fifth nucleotide sequences (R₁, R₂, R₃, R₄,R₅) encoding a first, second, third, fourth and fifth rotavirus protein(X₁, X₂, X₃, X₄, X₅) is expressed in a plant or portion of a plant (SeeTable 1, Combination #1.1).

Accordingly, nucleic acid N₁ comprises nucleotide sequences R₁, R₂, R₃,R₄ and R₅, wherein R₁ encodes rotavirus protein X₁, where X₁ may be anyrotavirus protein selected from the group of VP2, VP4, VP6, VP7 andNSP4, and wherein R₂-R₅ encode a rotavirus protein that is not X₁, whereX₂ may be any rotavirus protein selected from the group of VP2, VP4,VP6, VP7 and NSP4, and wherein R₁ and R₃-R₅ encode a rotavirus proteinthat is not X₂, where X₃ may be any rotavirus protein selected from thegroup of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁ R₂, R₄, and R₅encode a rotavirus protein that is not X₃, where X₄ may be any rotavirusprotein selected from the group of VP2, VP4, VP6, VP7 and NSP4, andwherein R₁-R₃ and R₅ encode a rotavirus protein that is not X₄, where X₅may be any rotavirus protein selected from the group of VP2, VP4, VP6,VP7 and NSP4, and wherein R₁-R₄ encode a rotavirus protein that is notX₅, with the result that a nucleotide sequence encoding for eachrotavirus protein VP2, VP4, VP6, VP7 and NSP4 is comprised on nucleicacid N₁, thereby allowing for the expression of each rotavirus proteinVP2, VP4, VP6, VP7 and NSP4 in the transformed host.

The nucleic acid may comprise a nucleotide sequence R₁, wherein R₁ mayencode rotavirus protein VP2, VP4, VP6, VP7 or NSP4 and nucleotidesequences R₂-R₅, wherein R₂-R₅ encode a rotavirus protein selected fromVP2, VP4, VP6, VP7 or NSP4, and wherein the rotavirus protein is notencoded by R₁. For example which is not to be considered limiting,nucleotide sequence R₁ may encode rotavirus protein VP2 and nucleotidesequence R₂-R₅ may encode in any order rotavirus protein VP4, VP6, VP7and NSP4, but R₂-R₅ may not encode VP2. In another example which is notto be considered limiting, nucleotide sequence R₁ may encode rotavirusprotein VP4 and nucleotide sequence R₂-R₅ may encode in any orderrotavirus protein VP2, VP6, VP7 and NSP4, but R₂-R₅ may not encode VP4.In yet another non-limiting example, nucleotide sequence R₁ may encoderotavirus protein VP6 and nucleotide sequence R₂-R₅ may encode in anyorder rotavirus protein VP2, VP4, VP7 and NSP4, but R₂-R₅ may not encodeVP6. In yet another example which is not to be considered limiting,nucleotide sequence R₁ may encode rotavirus protein VP7 and nucleotidesequence R₂-R₅ may encode in any order rotavirus protein VP2, VP4, VP6and NSP4, but R₂-R₅ may not encode VP7. In yet another example which isnot to be considered limiting, nucleotide sequence R₁ may encoderotavirus protein NSP4 and nucleotide sequence R₂-R₅ may encode in anyorder rotavirus protein VP2, VP4, VP6 and VP7, but R₂-R₅ may not encodeNSP4.

For example, which is not to be considered limiting, the nucleotidesequence (N₁) comprises a first nucleotide sequences (R₁) encoding afirst rotavirus protein, for example rotavirus protein VP7, a secondnucleotide sequences (R₂) encoding a second rotavirus protein, forexample rotavirus protein VP4, a third nucleotide sequences (R₃)encoding a third rotavirus protein, for example rotavirus protein NSP4,a fourth nucleotide sequences (R₄) encoding a fourth rotavirus protein,for example rotavirus protein VP6 and a fifth nucleotide sequences (R₅)encoding a fifth rotavirus protein, for example rotavirus protein VP2.

In a further non-limiting example the nucleotide sequence (N₁) comprisesa first nucleotide sequences (R₁) encoding a first rotavirus protein,for example rotavirus protein VP4, a second nucleotide sequences (R₂)encoding a second rotavirus protein, for example rotavirus protein VP7,a third nucleotide sequences (R₃) encoding a third rotavirus protein,for example rotavirus protein NSP4, a fourth nucleotide sequences (R₄)encoding a fourth rotavirus protein, for example rotavirus protein VP6and a fifth nucleotide sequences (R₅) encoding a fifth rotavirusprotein, for example rotavirus protein VP2.

In a further non-limiting example the nucleotide sequence (N₁) comprisesa first nucleotide sequences (R₁) encoding a first rotavirus protein,for example rotavirus protein VP4, a second nucleotide sequences (R₂)encoding a second rotavirus protein, for example rotavirus protein VP7,a third nucleotide sequences (R₃) encoding a third rotavirus protein,for example rotavirus protein VP6, a fourth nucleotide sequences (R₄)encoding a fourth rotavirus protein, for example rotavirus protein VP2and a fifth nucleotide sequences (R₅) encoding a fifth rotavirusprotein, for example rotavirus protein NSP4.

In another non-limiting example the nucleotide sequence (N₁) comprises afirst nucleotide sequences (R₁) encoding a first rotavirus protein, forexample rotavirus protein VP7, a second nucleotide sequences (R₂)encoding a second rotavirus protein, for example rotavirus protein VP4,a third nucleotide sequences (R₃) encoding a third rotavirus protein,for example rotavirus protein VP6, a fourth nucleotide sequences (R₄)encoding a fourth rotavirus protein, for example rotavirus protein VP2and a fifth nucleotide sequences (R₅) encoding a fifth rotavirusprotein, for example rotavirus protein NSP4. (see FIG. 5) A plant may betransformed with a single nucleic acid (N₁) comprising a first, second,third, fourth and fifth nucleotide sequences (R₁, R₂, R₃, R₄, R₅)encoding a first, second, third, fourth and fifth rotavirus protein, sothat each of the first, second, third, fourth and fifth protein areexpressed in the plant. The rotavirus proteins are selected from thegroup of rotavirus protein VP2, VP4, VP6, VP7 and NSP4, so that each ofVP2, VP4, VP6, VP7 and NSP4 are expressed in the plant. The singlenucleic acid may be introduced in the plant in a transient manner, or ina stable manner.

The VP4 may be processed or cleaved to produce VP5 and VP8 within thehost by co-expressing a nucleic acid encoding a suitable protease, forexample, trypsin, a trypsin-like protease, a serine protease, achymotrypsin-like protease, subtilisin. Alternatively, VP4 may beprocessed during any step of RLP extraction, or after RLP purificationby adding a sutible protease, for example, trypsin, a trypsin-likeprotease, a serine protease, a chymotrypsin-like protease, subtilisin.

1.2. Quadruple Gene Construct

As described herein, a method of producing RLPs in a plant is provided,wherein a nucleic acid (N₁; a first nucleic acid) comprising a first,second, third and fourth nucleotide sequences (R₁, R₂, R₃, R₄) encodinga first, second, third and fourth rotavirus protein (X₁, X₂, X₃, X₄) isexpressed in a plant or portion of a plant (See Table 1, Combination#1.2).

Accordingly, nucleic acid N₁ may comprises nucleotide sequences (R₁, R₂,R₃, R₄), wherein R₁ encodes rotavirus protein X₁, where X₁ may be anyrotavirus protein selected from the group of VP2, VP4, VP6 and NSP4, andwherein R₂-R₄ encode a rotavirus protein that is not X₁, where X₂ may beany rotavirus protein selected from the group of VP2, VP4, VP6 and NSP4,and wherein R₁ and R₃-R₄ encode a rotavirus protein that is not X₂,where X₃ may be any rotavirus protein selected from the group of VP2,VP4, VP6 and NSP4, and wherein R₁ R₂, and R₄ encode a rotavirus proteinthat is not X₃, where X₄ may be any rotavirus protein selected from thegroup of VP2, VP4, VP6 and NSP4, and wherein R₁-R₃ encode a rotavirusprotein that is not X₄, with the result that a nucleotide sequenceencoding for each rotavirus protein VP2, VP4, VP6 and NSP4 is comprisedon nucleic acid N₁, thereby allowing for the expression of eachrotavirus protein VP2, VP4, VP6 and NSP4 in the transformed host.

For example, which is not to be considered limiting, the nucleotidesequence (N₁) comprises a first nucleotide sequences (R₁) encoding afirst rotavirus protein, for example rotavirus protein VP6, a secondnucleotide sequences (R₂) encoding a second rotavirus protein, forexample rotavirus protein VP4, a third nucleotide sequences (R₃)encoding a third rotavirus protein, for example rotavirus proteinVP2,and a fourth nucleotide sequences (R₄) encoding a fourth rotavirusprotein, for example rotavirus protein NSP4.

Further accordingly, nucleic acid N₁ may comprises nucleotide sequences(R₁, R₂, R₃, R₄), wherein R₁ encodes rotavirus protein X₁, where X₁ maybe any rotavirus protein selected from the group of VP2, VP7, VP6 andNSP4, and wherein R₂-R₄ encode a rotavirus protein that is not X₁, whereX₂ may be any rotavirus protein selected from the group of VP2, VP7, VP6and NSP4, and wherein R₁ and R₃-R₄ encode a rotavirus protein that isnot X₂, where X₃ may be any rotavirus protein selected from the group ofVP2, VP7, VP6 and NSP4, and wherein R₁ R₂, and R₄ encode a rotavirusprotein that is not X₃, where X₄ may be any rotavirus protein selectedfrom the group of VP2, VP7, VP6 and NSP4, and wherein R₁-R₃ encode arotavirus protein that is not X₄, with the result that a nucleotidesequence encoding for each rotavirus protein VP2, VP7, VP6 and NSP4 iscomprised on nucleic acid N₁, thereby allowing for the expression ofeach rotavirus protein VP2, VP7, VP6 and NSP4 in the transformed host.

For example, which is not to be considered limiting, the nucleotidesequence (N₁) comprises a first nucleotide sequences (R₁) encoding afirst rotavirus protein, for example rotavirus protein VP6, a secondnucleotide sequences (R₂) encoding a second rotavirus protein, forexample rotavirus protein VP7, a third nucleotide sequences (R₃)encoding a third rotavirus protein, for example rotavirus proteinVP2,and a fourth nucleotide sequences (R₄) encoding a fourth rotavirusprotein, for example rotavirus protein NSP4.

Further accordingly, nucleic acid N₁ may comprises nucleotide sequences(R₁, R₂, R₃, R₄), wherein R₁ encodes rotavirus protein X₁, where X₁ maybe any rotavirus protein selected from the group of VP7, VP4, VP6 andNSP4, and wherein R₂-R₄ encode a rotavirus protein that is not X₁, whereX₂ may be any rotavirus protein selected from the group of VP7, VP4, VP6and NSP4, and wherein R₁ and R₃-R₄ encode a rotavirus protein that isnot X₂, where X₃ may be any rotavirus protein selected from the group ofVP7, VP4, VP6 and NSP4, and wherein R₁ R₂, and R₄ encode a rotavirusprotein that is not X₃, where X₄ may be any rotavirus protein selectedfrom the group of VP7, VP4, VP6 and NSP4, and wherein R₁-R₃ encode arotavirus protein that is not X₄, with the result that a nucleotidesequence encoding for each rotavirus protein VP7, VP4, VP6 and NSP4 iscomprised on nucleic acid N₁, thereby allowing for the expression ofeach rotavirus protein VP7, VP4, VP6 and NSP4 in the transformed host.

For example, which is not to be considered limiting, the nucleotidesequence (N₁) comprises a first nucleotide sequences (R₁) encoding afirst rotavirus protein, for example rotavirus protein VP6, a secondnucleotide sequences (R₂) encoding a second rotavirus protein, forexample rotavirus protein VP4, a third nucleotide sequences (R₃)encoding a third rotavirus protein, for example rotavirus proteinVP7,and a fourth nucleotide sequences (R₄) encoding a fourth rotavirusprotein, for example rotavirus protein NSP4.

1.3 Triple Gene Construct

As described herein, a method of producing RLPs in a plant is provided,wherein a nucleic acid (N₁; a first nucleic acid) comprising a first,second and third nucleotide sequences (R₁, R₂, R₃) encoding a first,second and third rotavirus protein (X₁, X₂, X₃) is expressed in a plantor portion of a plant (See Table 1, Combination #1.3).

Accordingly, nucleic acid N₁ may comprises nucleotide sequences (R₁, R₂,R₃), wherein R₁ encodes rotavirus protein X₁, where X₁ may be anyrotavirus protein selected from the group of VP4, VP6 and NSP4, andwherein R₂-R₃ encode a rotavirus protein that is not X₁, where X₂ may beany rotavirus protein selected from the group of VP4, VP6 and NSP4, andwherein R₁ and R₃ encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP4, VP6 andNSP4, and wherein R₁ R₂ encode a rotavirus protein that is not X₃, withthe result that a nucleotide sequence encoding for each rotavirusprotein VP4, VP6 and NSP4 is comprised on nucleic acid N₁, therebyallowing for the expression of each rotavirus protein VP4, VP6 and NSP4in the transformed host.

For example, which is not to be considered limiting, the nucleotidesequence (N₁) comprises a first nucleotide sequences (R₁) encoding afirst rotavirus protein, for example rotavirus protein VP6, a secondnucleotide sequences (R₂) encoding a second rotavirus protein, forexample rotavirus protein VP4, a third nucleotide sequences (R₃)encoding a third rotavirus protein, for example rotavirus protein.

Further accordingly, nucleic acid N₁ may comprises nucleotide sequences(R₁, R₂, R₃), wherein R₁ encodes rotavirus protein X₁, where X₁ may beany rotavirus protein selected from the group of VP7, VP6 and NSP4, andwherein R₂-R₃ encode a rotavirus protein that is not X₁, where X₂ may beany rotavirus protein selected from the group of VP7, VP6 and NSP4, andwherein R₁ and R₃ encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP7, VP6 andNSP4, and wherein R₁ R₂ encode a rotavirus protein that is not X₃, withthe result that a nucleotide sequence encoding for each rotavirusprotein VP7, VP6 and NSP4 is comprised on nucleic acid N₁, therebyallowing for the expression of each rotavirus protein VP7, VP6 and NSP4in the transformed host.

For example, which is not to be considered limiting, the nucleotidesequence (N₁) comprises a first nucleotide sequences (R₁) encoding afirst rotavirus protein, for example rotavirus protein VP6, a secondnucleotide sequences (R₂) encoding a second rotavirus protein, forexample rotavirus protein VP7, a third nucleotide sequences (R₃)encoding a third rotavirus protein, for example rotavirus protein.

2. Two Constructs

2.1. Quadruple Gene Construct+Single Gene Construct

The present invention also provides for a method of producing RLPs in aplant, wherein a first nucleic acid (N₁) comprising a nucleotidesequence (R₁) encoding a first rotavirus protein (X₁), is co-expressedwith a second nucleic acid (N₂) comprising four nucleotide sequences(R₂-R₅) encoding a second, third, fourth and fifth rotavirus protein(X₂-X₅) (see Table 1, Combination #2.1), so that the first, second,third, fourth and fifth nucleotide sequence (R₁-R₅) are co-expressed inthe plant.

In this non-limiting example, N₁ comprises nucleotide sequence (R₁) andN₂ comprises nucleotide sequences (R₂, R₃, R₄, R₅), wherein eachrotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4is encoded in the combination of both constructs N₁ and N₂, and whereinR₁ encodes rotavirus protein X₁, wherein X₁ may be any rotavirus proteinselected from the group of VP2, VP4, VP6, VP7 and NSP4, and whereinR₂-R₅ encode a rotavirus protein that is not X₁, where X₂ may be anyrotavirus protein selected from the group of VP2, VP4, VP6, VP7 andNSP4, and wherein R₁ and R₃-R₅ encode a rotavirus protein that is notX₂, where X₃ may be any rotavirus protein selected from the group ofVP2, VP4, VP6, VP7 and NSP4, and wherein R₁ R₂, R₄, and R₅ encode arotavirus protein that is not X₃, where X₄ may be any rotavirus proteinselected from the group of VP2, VP4, VP6, VP7 and NSP4, and whereinR₁-R₃ and R₅ encode a rotavirus protein that is not X₄, where X₅ may beany rotavirus protein selected from the group of VP2, VP4, VP6, VP7 andNSP4, and wherein R₁-R₄ encode a rotavirus protein that is not X₅, withthe result that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 isexpressed in the host.

For example, which is not to be considered limiting, nucleotide sequenceR₁ may encode rotavirus protein VP2 and nucleotide sequence R₂-R₅ mayencode in any order rotavirus protein VP4, VP6, VP7 and NSP4, but R₂-R₅may not encode VP2. In another non-limiting example nucleotide sequenceR₁ may encode rotavirus protein VP4 and nucleotide sequence R₂-R₅ mayencode in any order rotavirus protein VP2, VP6, VP7 and NSP4, but R₂-R₅may not encode VP4. In yet another non-limiting example nucleotidesequence R₁ may encode rotavirus protein VP6 and nucleotide sequenceR₂-R₅ may encode in any order rotavirus protein VP2, VP4, VP7 and NSP4,but R₂-R₅ may not encode VP6. In yet another example which is not to beconsidered limiting, nucleotide sequence R₁ may encode rotavirus proteinVP7 and nucleotide sequence R₂-R₅ may encode in any order rotavirusprotein VP2, VP4, VP6 and NSP4, but R₂-R₅ may not encode VP7. In yetanother non-limiting example nucleotide sequence R₁ may encode rotavirusprotein NSP4 and nucleotide sequence R₂-R₅ may encode in any orderrotavirus protein VP2, VP4, VP6 and VP7, but R₂-R₅ may not encode NSP4.

The first nucleic acid (N₁) and second nucleic acid (N₂) may beintroduced into the plant in the same step, or may be introduced to theplant sequentially.

For example, which is not to be considered limiting, a first nucleotidesequence (N₁) comprising a nucleotide sequence (R₁) encoding a firstrotavirus protein, for example NSP4, is co-expressed with a secondnucleic acid (N₂) comprising four nucleotide sequences (R₂-R₅) encodinga second, third, fourth and fifth rotavirus protein, for example VP7,VP4, VP6 and VP2, respectively (see FIG. 5).

In another non-limiting example, a first nucleotide sequence (N₁)comprising a nucleotide sequence (R₁) encoding a first rotavirusprotein, for example NSP4, is co-expressed with a second nucleic acid(N₂) comprising four sequential nucleotide sequences (R₂-R₅) encoding asecond, third, fourth and fifth rotavirus protein, for example VP4, VP7,VP6 and VP2, respectively. In yet another non-limiting example, a firstnucleotide sequence (N₁) comprising a nucleotide sequence (R₁) encodinga first rotavirus protein, for example NSP4, is co-expressed with asecond nucleic acid (N₂) comprising four sequential nucleotide sequences(R₂-R₅) encoding a second, third, fourth and fifth rotavirus protein,for example VP7, VP4, VP2 and VP6, respectively. In yet anothernon-limiting example, a first nucleotide sequence (N₁) comprising anucleotide sequence (R₁) encoding a first rotavirus protein, for exampleNSP4, is co-expressed with a second nucleic acid (N₂) comprising foursequential nucleotide sequences (R₂-R₅) encoding a second, third, fourthand fifth rotavirus protein, for example VP4, VP7, VP2 and VP6,respectively. In yet another non-limiting example, a first nucleotidesequence (N₁) comprising a nucleotide sequence (R₁) encoding a firstrotavirus protein, for example NSP4, is co-expressed with a secondnucleic acid (N₂) comprising four sequential nucleotide sequences(R₂-R₅) encoding a second, third, fourth and fifth rotavirus protein,for example VP6, VP2, VP4 and VP7, respectively. In yet anothernon-limiting example, a first nucleotide sequence (N₁) comprising anucleotide sequence (R₁) encoding a first rotavirus protein, for exampleNSP4, is co-expressed with a second nucleic acid (N₂) comprising foursequential nucleotide sequences (R₂-R₅) encoding a second, third, fourthand fifth rotavirus protein, for example VP6, VP2, VP7 and VP4,respectively. In yet another non-limiting example, a first nucleotidesequence (N₁) comprising a nucleotide sequence (R₁) encoding a firstrotavirus protein, for example NSP4, is co-expressed with a secondnucleic acid (N₂) comprising four sequential nucleotide sequences(R₂-R₅) encoding a second, third, fourth and fifth rotavirus protein,for example VP2, VP4, VP6 and VP7, respectively. In yet anothernon-limiting example, a first nucleotide sequence (N₁) comprising anucleotide sequence (R₁) encoding a first rotavirus protein, for exampleNSP4, is co-expressed with a second nucleic acid (N₂) comprising foursequential nucleotide sequences (R₂-R₅) encoding a second, third, fourthand fifth rotavirus protein, for example VP7, VP6, VP4 and VP2,respectively. In yet another non-limiting example, a first nucleotidesequence (N₁) comprising a nucleotide sequence (R₁) encoding a firstrotavirus protein, for example VP7, is co-expressed with a secondnucleic acid (N₂) comprising four sequential nucleotide sequences(R₂-R₅) encoding a second, third, fourth and fifth rotavirus protein,for example NSP4, VP2, VP6 and VP4, respectively. In yet anothernon-limiting example, a first nucleotide sequence (N₁) comprising anucleotide sequence (R₁) encoding a first rotavirus protein, for exampleVP4, is co-expressed with a second nucleic acid (N₂) comprising foursequential nucleotide sequences (R₂-R₅) encoding a second, third, fourthand fifth rotavirus protein, for example NSP4, VP2, VP6 and VP7,respectively.

A plant that expresses a first nucleic acid (N₁) comprising a firstnucleotide sequence (R₁) encoding a first rotavirus protein (X₁), may betransformed with a second nucleic acid (N₂) comprising four nucleotidesequences (R₂-R₅) encoding a second, third, fourth and fifth rotavirusprotein (X₂-X₅), so that the first, second, third, fourth and fifthnucleotide sequence are co-expressed in the plant. Furthermore, a plantthat expresses a first nucleic acid (N₂) comprising four nucleotidesequences (R₂-R₅) encoding a rotavirus protein X₂-X₅ may be transformedwith a second nucleic acid (N₁) comprising a nucleotide sequence (R₁)encoding a rotavirus protein (X₁), so that the first and secondnucleotide sequences R₁-R₅ are co-expressed in the plant. The rotavirusprotein X₁ may be any rotavirus protein selected from the group of VP2,VP4, VP6, VP7 and NSP4, and rotavirus proteins X₂-X₅ may be anyrotavirus protein selected from the group of VP2, VP₄, VP6, VP7 andNSP4, but not X₁, so that each rotavirus protein VP2, VP4, VP₆, VP7 andNSP4 is expressed. Furthermore, a plant may be simultaneouslyco-transformed with a first nucleic acid (N₂) comprising four nucleotidesequences (R₂-_(R5)) encoding a rotavirus protein X₂-X₅, and with asecond nucleic acid (N₁) comprising a nucleotide sequence (R₁) encodinga rotavirus protein (X₁), so that the first and second nucleotidesequences R₁-R₅ are co-expressed in the plant. The rotavirus protein X₁may be any rotavirus protein selected from the group of VP2, VP4, VP6,VP7 and NSP4, and rotavirus proteins X₂-X₅ may be any rotavirus proteinselected from the group of VP2, VP4, VP6, VP7 and NSP4, but not theprotein selected for X₁, so that each rotavirus protein VP2, VP4, VP6,VP7 and NSP4 is expressed. The first nucleic acid (N₁) and secondnucleic acid (N₂) may be introduced in the plant in a transient manner,or in a stable manner.

For example, which is not to be considered limiting, a plant thatexpresses a first nucleic acid (N₁) comprising a first nucleotidesequence (R₁) encoding a first rotavirus protein (X₁) for example NSP4,may be transformed with a second nucleic acid encoding (N₂) comprisingfour nucleotide sequences (R₂-R₅) encoding a second, third, fourth andfifth rotavirus protein (X₂-X₅), for example VP7, VP4, VP6 and VP2, sothat NSP4, VP7, VP4, VP6 and VP2 are co-expressed in the plant.

A first plant expressing a first nucleic acid (N₁) comprising a firstnucleotide sequence (R₁) encoding a first rotavirus protein (X₁), may becrossed with a second plant expressing the second nucleic acid (N₂)comprising four nucleotide sequences (R₂-R₅) encoding a second, third,fourth and fifth rotavirus protein (X₂-X₅) to produce a progeny plant(third plant) that co-expresses the first, second, third, fourth andfifth rotavirus protein (X₁-X₅). Furthermore, a first plant expressing afirst nucleic acid (N₂) comprising four nucleotide sequences (R₂-R₅)encoding a rotavirus protein X₂-X₅ may be crossed with a second plantexpressing a second nucleic acid (N₁) comprising a nucleotide sequence(R₁) encoding a rotavirus protein (X₁), so that nucleotide sequencesR₁-R₅ are co-expressed in the progeny plant. The rotavirus protein maybe any rotavirus protein selected from the group of rotavirus proteinVP2, VP4, VP6, VP7 and NSP4, wherein VP2, VP4, VP6, VP7 and NSP4 areselected once, so that each rotavirus protein VP2, VP4, VP6, VP7 andNSP4 is expressed in the progeny plant.

The VP4 may be processed or cleaved to produce VP5 and VP8 within thehost by co-expressing a nucleic acid encoding a suitable protease, forexample, trypsin, a trypsin-like protease, a serine protease, achymotrypsin-like protease, subtilisin. Alternatively, VP4 may beprocessed during any step of RLP extraction, or after RLP purificationby adding a suitable protease, for example, trypsin, a trypsin-likeprotease, a serine protease, a chymotrypsin-like protease, subtilisin.

2.2 Triple Gene Construct+Dual Gene Construct

The present invention also provides for a method of producing RLPs in aplant, wherein a first nucleic acid (N₁) comprising two nucleotidesequences (R₁ and R₂) encoding a first rotavirus protein (X₁) and secondrotavirus protein (X₂) respectively, is co-expressed with a secondnucleic acid (N₂) comprising three nucleotide sequences (R₃-R₅) encodinga third, fourth and fifth rotavirus proteins (X₃-X₅) (see Table 1,Combination #2.2), so that the first, second, third, fourth and fifthnucleotide sequence are co-expressed in the plant.

In a non-limiting example, N₁ comprises nucleotide sequences (R₁, R₂)and N₂ comprises nucleotide sequences (R₃, R₄, R₅), wherein eachrotavirus protein selected from the group of VP2, VP4, VP6, V_(P7) andNSP4 is encoded and wherein R₁ encodes rotavirus protein X₁, wherein X₁may be any rotavirus protein selected from the group of VP2, VP4, VP6,VP7 and NSP4, and wherein R₂-R₅ encode a rotavirus protein that is notX₁, where X₂ may be any rotavirus protein selected from the group ofVP2, VP4, VP6, VP7 and NSP4, and wherein R₁ and R₃-R₅ encode a rotavirusprotein that is not X₂, where X₃ may be any rotavirus protein selectedfrom the group of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁ R₂, R₄,and R₅ encode a rotavirus protein that is not X₃, where X₄ may be anyrotavirus protein selected from the group of VP2, VP4, VP6, VP7 andNSP4, and wherein R₁-R₃ and R₅ encode a rotavirus protein that is notX₄, where X₅ may be any rotavirus protein selected from the group ofVP2, VP4, VP6, VP7 and NSP4, and wherein R₁-R₄ encode a rotavirusprotein that is not X₅, with the result that each rotavirus protein VP2,VP4, VP6, VP7 and NSP4 is expressed in the host.

Therefore, the first nucleic acid (N₁) may comprise a nucleotidesequence R₁, wherein R₁ may encode rotavirus protein VP2, VP4, VP6, VP7or NSP4 and a second nucleotide sequence R₂ which may encode anyrotavirus protein selected from the group of VP2, VP4, VP6, VP7 andNSP4, that is not encoded by R₁. The second nucleic acid (N₂) maycomprise nucleotide sequences R₃-R₅, wherein R₃-R₅ encode rotavirusprotein VP2, VP4, VP6, VP7 or NSP4, but not rotavirus protein that areencoded by R₁ or R₂. For example, which is not to be consideredlimiting, nucleotide sequence R₁ may encode rotavirus protein VP2 andnucleotide sequence R₂-R₅ may encode in any order rotavirus protein VP4,VP6, VP7 and NSP4, but R₂-R₅ may not encode VP2. In another non-limitingexample nucleotide sequence R₁ may encode rotavirus protein VP4 andnucleotide sequences R₂-R₅ may encode in any order rotavirus proteinVP2, VP6, VP7 and NSP4. In yet another example nucleotide sequence R₁may encode rotavirus protein VP6 and nucleotide sequences R₂-R₅ mayencode in any order rotavirus protein VP2, VP4, VP7 and NSP4. In yetanother non-limiting example nucleotide sequence R₁ may encode rotavirusprotein VP7 and nucleotide sequences R₂-R₅ may encode in any orderrotavirus protein VP2, VP4, VP6 and NSP4. In yet another non-limitingexample nucleotide sequence R₁ may encode rotavirus protein NSP4 andnucleotide sequences R₂-R₅ may encode in any order rotavirus proteinVP2, VP4, VP6 and VP7.

A plant that expresses a first nucleic acid (N₁) comprising a first andsecond nucleotide sequence (R₁+R₂) encoding a first and second rotavirusprotein (X₁+X₂), may be transformed with a second nucleic acid (N₂)comprising three nucleotide sequences (R₃-R₅) encoding a third, fourthand fifth rotavirus protein (X₃-X₅), so that the first, second, third,fourth and fifth nucleotide sequence are co-expressed in the plant. Thefirst nucleic acid (N₁) and second nucleic acid (N₂) may be introducedin the plant in a transient manner, or in a stable manner.

Furthermore, a plant that expresses a first nucleic acid (N₂) comprisingthree nucleotide sequences (R₃-R₅) encoding a first, second and thirdrotavirus protein (X₃-X₅) may be transformed with a second nucleic acid(N₁) comprising a fourth nucleotide sequence (R₁) encoding a fourthrotavirus protein (X₁) and a fifth rotavirus protein (X₂) so that thefirst and second nucleic acids R₁-R₅ are co-expressed in the plant. Therotavirus protein X₁ may be any rotavirus protein selected from thegroup of VP2, VP4, VP6, VP7 and NSP4, and rotavirus proteins X₂-X₅ maybe any rotavirus protein selected from the group of VP2, VP4, VP6, VP7and NSP4, but not X₁, so that each rotavirus protein VP2, VP4, VP6, VP7and NSP4 is expressed. For example, which is not to be consideredlimiting, a plant that expresses a first nucleic acid (N₂) comprisingthree nucleotide sequences (R₃-R₅) encoding a first, second and thirdrotavirus proteins (X₃-X₅), for example VP7, VP4 and VP6 may betransformed with a second nucleic acid encoding (N₁) comprising a fourthand a fifth nucleotide sequences (R₁-R₂) encoding a fourth and a fifthrotavirus protein (X₁-X₂) for example VP2 and NSP4, so that NSP4, VP7,VP4, VP6 and VP2 are co-expressed in the plant. Furthermore, a plant maybe simultaneously co-transformed with a first nucleic acid (N₁)comprising a first and second nucleotide sequence (R₁+R₂) encoding afirst and second rotavirus protein (X₁+X₂), and a second nucleic acid(N₂) comprising three nucleotide sequences (R₃-R₅) encoding a third,fourth and fifth rotavirus protein (X₃-X₅), so that the first, second,third, fourth and fifth nucleotide sequence are co-expressed in theplant. The first nucleic acid (N₁) and second nucleic acid (N₂) may beintroduced in the plant in a transient manner, or in a stable manner.

A first plant expressing a first nucleic acid (N₁) comprising a firstnucleotide sequence (R₁) encoding a first rotavirus protein (X₁) and asecond nucleotide sequence (R₂) encoding a second rotavirus protein(X₂), may be crossed with a second plant expressing the second nucleicacid (N₂) comprising three nucleotide sequences (R₃-R₅) encoding athird, fourth and fifth rotavirus protein (X₃-X₅) to produce a progenyplant (third plant) that co-expresses the first, second, third, fourthand fifth rotavirus protein (X₁-X₅). Furthermore, a first plantexpressing a first nucleic acid (N₂) comprising three nucleotidesequences (R₃-R₅) encoding a rotavirus protein X₃-X₅ may be crossed witha second plant expressing a second nucleic acid (N₁) comprising anucleotide sequence (R₁) encoding a rotavirus protein (X₁) and a secondnucleotide sequence (R₂) encoding a second rotavirus protein (X₂), sothat nucleotide sequences R₁-R₅ are co-expressed in the progeny plant.The rotavirus protein may be any rotavirus protein selected from thegroup of rotavirus protein VP2, VP4, VP6, VP7 and NSP4, wherein VP2,VP4, VP6, VP7 and NSP4 are selected once, so that each rotavirus proteinVP2, VP4, VP6, VP7 and NSP4 is expressed in the progeny plant.

2.3. Triple Gene Construct+Single Gene Construct

The present invention also provides for a method of producing RLPs in aplant, wherein a first nucleic acid (N₁) comprising one nucleotidesequence (R₁) encoding a first rotavirus protein (X₁), is co-expressedwith a second nucleic acid (N₂) comprising three nucleotide sequences(R₂-R₄) encoding a second, third and fourth rotavirus proteins (X₂-X₄),so that the first, second, third and fourth nucleotide sequence areco-expressed in the plant (See Table 1, Combination #2.3).

In a non-limiting example, N₁ comprises nucleotide sequence (R₁) and N₂comprises nucleotide sequences (R₂, R₃, R₄), wherein each rotavirusprotein selected from the group of VP2, VP4, VP6 and NSP4 is encoded andwherein R₁ encodes rotavirus protein X₁, wherein X₁ may be any rotavirusprotein selected from the group of VP2, VP4, VP6 and NSP4, and whereinR₂-R₄ encode a rotavirus protein that is not X₁, where X₂ may be anyrotavirus protein selected from the group of VP2, VP4, VP6 and NSP4, andwherein R₁ and R₃-R₄ encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP2, VP4, VP6and NSP4, and wherein R₁ R₂, and R₄ encode a rotavirus protein that isnot X₃, where X₄ may be any rotavirus protein selected from the group ofVP2, VP4, VP6 and NSP4, and wherein R₁-R₃ encode a rotavirus proteinthat is not X₄, with the result that each rotavirus protein VP2, VP4,VP6 and NSP4 is expressed in the host.

In a further non-limiting example, N₁ comprises nucleotide sequence (R₁)and N₂ comprises nucleotide sequences (R₂, R₃, R₄), wherein eachrotavirus protein selected from the group of VP2, VP7, VP6 and NSP4 isencoded and wherein R₁ encodes rotavirus protein X₁, wherein X₁ may beany rotavirus protein selected from the group of VP2, VP7, VP6 and NSP4,and wherein R₂-R₄ encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP2, VP7, VP6and NSP4, and wherein R₁ and R₃-R₄ encode a rotavirus protein that isnot X₂, where X₃ may be any rotavirus protein selected from the group ofVP2, VP7, VP6 and NSP4, and wherein R₁ R₂, and R₄ encode a rotavirusprotein that is not X₃, where X₄ may be any rotavirus protein selectedfrom the group of VP2, VP7, VP6 and NSP4, and wherein R₁-R₃ encode arotavirus protein that is not X₄, with the result that each rotavirusprotein VP2, VP7, VP6 and NSP4 is expressed in the host.

In yet a further non-limiting example, N₁ comprises nucleotide sequence(R₁) and N₂ comprises nucleotide sequences (R₂, R₃, R₄), wherein eachrotavirus protein selected from the group of VP4, VP7, VP6 and NSP4 isencoded and wherein R₁ encodes rotavirus protein X₁, wherein X₁ may beany rotavirus protein selected from the group of VP4, VP7, VP6 and NSP4,and wherein R₂-R₄ encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP4, VP7, VP6and NSP4, and wherein R₁ and R₃-R₄ encode a rotavirus protein that isnot X₂, where X₃ may be any rotavirus protein selected from the group ofVP4, VP7, VP6 and NSP4, and wherein R₁ R₂, and R₄ encode a rotavirusprotein that is not X₃, where X₄ may be any rotavirus protein selectedfrom the group of VP4, VP7, VP6 and NSP4, and wherein R₁-R₃ encode arotavirus protein that is not X₄, with the result that each rotavirusprotein VP4, VP7, VP6 and NSP4 is expressed in the host.

2.4 Two Double Gene Constructs

The present invention also provides for a method of producing RLPs in aplant, wherein a first nucleic acid (N₁) comprising nucleotide sequences(R₁-R₂) encoding a first and a second rotavirus proteins (X₁-X₂), isco-expressed with a second nucleic acid (N₂) comprising two nucleotidesequences (R₃-R₄) encoding a third and fourth rotavirus proteins(X₃-X₄), so that the first, second, third and fourth nucleotidesequences are co-expressed in the plant (See Table 1, Combination #2.4).

In a non-limiting example, N₁ comprises nucleotide sequences (R₁, R₂)and N₂ comprises nucleotide sequences (R₃, R₄), wherein each rotavirusprotein selected from the group of VP2, VP4, VP6 and NSP4 is encoded andwherein R₁ encodes rotavirus protein X₁, wherein X₁ may be any rotavirusprotein selected from the group of VP2, VP4, VP6 and NSP4, and whereinR₂-R₄ encode a rotavirus protein that is not X₁, where X₂ may be anyrotavirus protein selected from the group of VP2, VP4, VP6 and NSP4, andwherein R₁ and R₃-R₄ encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP2, VP4, VP6and NSP4, and wherein R₁ R₂, and R₄ encode a rotavirus protein that isnot X₃, where X₄ may be any rotavirus protein selected from the group ofVP2, VP4, VP6 and NSP4, and wherein R₁-R₃ encode a rotavirus proteinthat is not X₄, with the result that each rotavirus protein VP2, VP4,VP6 and NSP4 is expressed in the host.

In a further non-limiting example, N₁ comprises nucleotide sequences(R₁, R₂) and N₂ comprises nucleotide sequences (R₃, R₄), wherein eachrotavirus protein selected from the group of VP2, VP7, VP6 and NSP4 isencoded and wherein R₁ encodes rotavirus protein X₁, wherein X₁ may beany rotavirus protein selected from the group of VP2, VP7, VP6 and NSP4,and wherein R₂-R₄ encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP2, VP7, VP6and NSP4, and wherein R₁ and R₃-R₄ encode a rotavirus protein that isnot X₂, where X₃ may be any rotavirus protein selected from the group ofVP2, VP7, VP6 and NSP4, and wherein R₁ R₂, and R₄ encode a rotavirusprotein that is not X₃, where X₄ may be any rotavirus protein selectedfrom the group of VP2, VP7, VP6 and NSP4, and wherein R₁-R₃ encode arotavirus protein that is not X₄, with the result that each rotavirusprotein VP2, VP7, VP6 and NSP4 is expressed in the host.

In yet a further non-limiting example, N₁ comprises nucleotide sequences(R₁, R₂) and N₂ comprises nucleotide sequences (R₃, R₄), wherein eachrotavirus protein selected from the group of VP4, VP7, VP6 and NSP4 isencoded and wherein R₁ encodes rotavirus protein X₁, wherein X₁ may beany rotavirus protein selected from the group of VP4, VP7, VP6 and NSP4,and wherein R₂-R₄ encode a rotavirus protein that is not X₁, where X₂may be any rotavirus protein selected from the group of VP4, VP7, VP6and NSP4, and wherein R₁ and R₃-R₄ encode a rotavirus protein that isnot X₂, where X₃ may be any rotavirus protein selected from the group ofVP4, VP7, VP6 and NSP4, and wherein R₁ R₂, and R₄ encode a rotavirusprotein that is not X₃, where X₄ may be any rotavirus protein selectedfrom the group of VP4, VP7, VP6 and NSP4, and wherein R₁-R₃ encode arotavirus protein that is not X₄, with the result that each rotavirusprotein VP4, VP7, VP6 and NSP4 is expressed in the host.

2.5 Double Gene Construct+Single Gene Construct

The present invention also provides for a method of producing RLPs in aplant, wherein a first nucleic acid (N₁) comprising nucleotide sequences(R₁-R₂) encoding a first and a second rotavirus proteins (X₁-X₂), isco-expressed with a second nucleic acid (N₂) comprising a nucleotidesequence (R₃) encoding a third rotavirus proteins (X₃), so that thefirst, second and third nucleotide sequences are co-expressed in theplant (See Table 1, Combination #2.5).

In a non-limiting example, N₁ comprises nucleotide sequences (R₁, R₂)and N₂ comprises nucleotide sequence (R₃), wherein each rotavirusprotein selected from the group of VP4, VP6 and NSP4 is encoded andwherein R₁ encodes rotavirus protein X₁, wherein X₁ may be any rotavirusprotein selected from the group of VP4, VP6 and NSP4, and wherein R₂-R₃encode a rotavirus protein that is not X₁, where X₂ may be any rotavirusprotein selected from the group of VP4, VP6 and NSP4, and wherein R₁ andR₃ encode a rotavirus protein that is not X₂, where X₃ may be anyrotavirus protein selected from the group of VP4, VP6 and NSP4, andwherein R₁ and R₂ encode a rotavirus protein that is not X₃, with theresult that each rotavirus protein VP4, VP6 and NSP4 is expressed in thehost.

In a further non-limiting example, N₁ comprises nucleotide sequences(R₁, R₂) and N₂ comprises nucleotide sequence (R₃), wherein eachrotavirus protein selected from the group of VP7, VP6 and NSP4 isencoded and wherein R₁ encodes rotavirus protein X₁, wherein X₁ may beany rotavirus protein selected from the group of VP7, VP6 and NSP4, andwherein R₂-R₃ encode a rotavirus protein that is not X₁, where X₂ may beany rotavirus protein selected from the group of VP7, VP6 and NSP4, andwherein R₁ and R₃ encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP7, VP6 andNSP4, and wherein R₁ and R₂ encode a rotavirus protein that is not X₃,with the result that each rotavirus protein VP7, VP6 and NSP4 isexpressed in the host.

3. Three Constructs

3.1 Two Dual Gene Constructs+One Single Gene Construct

The present invention also provides for a method of producing RLPs in aplant, wherein a first nucleic acid (N₁) comprising a nucleotidesequence (R₁) encoding a first rotavirus protein (X₁) and a secondnucleotide sequence (R₂) encoding a second rotavirus protein (X₂), isco-expressed with a second nucleic acid (N₂) comprising a thirdnucleotide sequence (R₃) encoding a third rotavirus protein (X₃) and afourth nucleotide sequences (R₄) encoding a fourth rotavirus protein(X₄) and a third nucleic acid (N₃) comprising a fifth nucleotidesequence (R₅) encoding a fifth rotavirus protein (X₅) (see Table 1,Combination #3.1) so that the first, second, third, fourth and fifthnucleotide sequence are co-expressed in the plant.

In this non-limiting example, N₁ comprises (R₁, R₂), N₂ comprises (R₃,R₄) and N₃ comprises (R₅), wherein each rotavirus protein selected fromthe group of VP2, VP4, VP6, VP7 and NSP4 is encoded once and wherein R₁encodes rotavirus protein X₁, wherein X₁ may be any rotavirus proteinselected from the group of VP2, VP4, VP6, VP7 and NSP4, and whereinR₂-R₅ encode a rotavirus protein that is not X₁, where X₂ may be anyrotavirus protein selected from the group of VP2, VP4, VP6, VP7 andNSP4, and wherein R₁ and R₃-R₅ encode a rotavirus protein that is notX₂, where X₃ may be any rotavirus protein selected from the group ofVP2, VP4, VP6, VP7 and NSP4, and wherein R₁ R₂, R₄, and R₅ encode arotavirus protein that is not X₃, where X₄ may be any rotavirus proteinselected from the group of VP2, VP4, VP6, VP7 and NSP4, and whereinR₁-R₃ and R₅ encode a rotavirus protein that is not X₄, where X₅ may beany rotavirus protein selected from the group of VP2, VP4, VP6, VP7 andNSP4, and wherein R₁-R₄ encode a rotavirus protein that is not X₅, withthe result that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 isexpressed in the host.

For example, the first nucleic acid (N₁) may comprise a nucleotidesequence R₁, wherein R₁ may encode rotavirus protein VP2, VP4, VP6, VP7or NSP4 and a second nucleotide sequence R₂ which may encode anyrotavirus protein selected from the group of VP2, VP4, VP6, VP7 and NSP4that is not encoded by R₁. The second nucleotide sequence may comprisenucleotide sequences R₃ and R₄, wherein R₃ encode rotavirus protein VP2,VP4, VP6, VP7 or NSP4, but not a rotavirus protein that are encoded byR₁ or R₂, and R₄ encodes rotavirus protein VP2, VP4, VP6, VP7 or NSP4,but not a rotavirus protein that are encoded by R₁, R₂ or R₃. The thirdnucleotide sequence may comprise nucleotide sequences R₅, wherein R₅encode rotavirus protein VP2, VP4, VP6, VP7 or NSP4, but not a rotavirusprotein that are encoded by R₁, R₂, R₂ or R₄, so that each of VP2, VP4,VP6, VP7 or NSP4 are expressed in a host.

For example which is not to be considered limiting, nucleotide sequenceR₁ may encode rotavirus protein VP2 and nucleotide sequence R₂-R₅ mayencode in any order rotavirus protein VP4, VP6, VP7 and NSP4, but R₂-R₅may not encode VP2. In another example nucleotide sequence R₁ may encoderotavirus protein VP4 and nucleotide sequences R₂-R₅ may encode in anyorder rotavirus protein VP2, VP6, VP7 and NSP4. In yet another examplenucleotide sequence R₁ may encode rotavirus protein VP6 and nucleotidesequences R₂-R₅ may encode in any order rotavirus protein VP2, VP4, VP7and NSP4. In yet another non-limiting example nucleotide sequence R₁ mayencode rotavirus protein VP7 and nucleotide sequences R₂-R₅ may encodein any order rotavirus protein VP2, VP4, VP6 and NSP4. In yet anothernon-limiting example nucleotide sequence R₁ may encode rotavirus proteinNSP4 and nucleotide sequences R₂-R₅ may encode in any order rotavirusprotein VP2, VP4, VP6 and VP7.

For example, which is not to be considered limiting, a first nucleicacid (N₁) comprising a first nucleotide sequence (R₁) encoding a firstrotavirus protein, for example VP6 and a second nucleotide sequence (R₂)encoding a second rotavirus protein, for example VP2, is co-expressedwith a second nucleic acid (N₂) comprising a third nucleotide sequence(R₃) encoding a third rotavirus protein for example VP7 and a fourthnucleotide sequence (R₄) encoding a fourth rotavirus protein, forexample VP4 and a third nucleic acid (N₃) comprising a fifth nucleotidesequence (R₅) encoding a fifth rotavirus protein for example NSP4. (seeFIGS. 2 and 3).

A plant that expresses a first nucleic acid (N₁) comprising a firstnucleotide sequence (R₁) encoding a first rotavirus protein (X₁) and asecond rotavirus protein (X₂), may be transformed with a second nucleicacid (N₂) comprising a third nucleotide sequence (R₃) encoding a thirdrotavirus protein (X₃) and a fourth nucleotide sequences (R₄) encoding afourth rotavirus protein (X₄). The plant may be further transformed witha third nucleic acid (R₃) comprising a fifth nucleotide sequence (R₅)encoding a fifth rotavirus protein (X₅), so that the first, second,third, fourth and fifth nucleotide sequence are co-expressed in theplant. Rotavirus protein X₁-X₅ may be selected from the group of VP2,VP4, VP6, VP7 and NSP4, wherein VP2, VP4, VP6, VP7 and NSP4 are selectedonce, so that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 isexpressed in the plant. Furthermore, a plant may be simultaneouslyco-transformed with a first nucleic acid (N₁) comprising a first andsecond nucleotide sequence (R₁+R₂) encoding a first and second rotavirusprotein (X₁+X₂), a second nucleic acid (N₂) comprising a third and afourth nucleotide sequences (R₃+R₄) encoding a third and a fourthrotavirus protein (X₃+X₄), and a third nucleic acid (N₃) comprising afifth nucleotide sequences (R₅) encoding a fifth rotavirus protein (X₅),so that the first, second, third, fourth and fifth nucleotide sequenceare co-expressed in the plant. The first nucleic acid (N₁), secondnucleic acid (N₂) and third nucleic acid (N₃) may be introduced in theplant in a transient manner, or in a stable manner.

3.2 Two Single Gene Constructs+One Triple Gene Construct

An alternated method of producing RLPs in a plant is also provided,wherein a first nucleic acid (N₁) comprising a nucleotide sequence (R₁)encoding a first rotavirus protein (X₁) is co-expressed with a secondnucleic acid (N₂) comprising a second nucleotide sequence (R₂) encodinga second rotavirus protein (X₂) and a third nucleic acid (N₃) comprisinga third nucleotide sequence (R₃) encoding a third rotavirus protein(X₃), a fourth nucleotide sequences (R₄) encoding a fourth rotavirusprotein (X₄) and a fifth nucleotide sequence (R₅) encoding a fifthrotavirus protein (X₅) (see Table 1, Combination #3.2) so that thefirst, second, third, fourth and fifth nucleotide sequence areco-expressed in the plant.

In an alternate example, N₁ comprises (R₁), N₂ comprises (R₂) and N₃comprises (R₃, R₄, R₅), wherein each rotavirus protein selected from thegroup of VP2, VP4, VP6, VP7 and NSP4 is encoded once and wherein R₁encodes rotavirus protein X₁, wherein X₁ may be any rotavirus proteinselected from the group of VP2, VP4, VP6, VP7 and NSP4, and whereinR₂-R₅ encode a rotavirus protein that is not X₁, where X₂ may be anyrotavirus protein selected from the group of VP2, VP4, VP6, VP7 andNSP4, and wherein R₁ and R₃-R₅ encode a rotavirus protein that is notX₂, where X₃ may be any rotavirus protein selected from the group ofVP2, VP4, VP6, VP7 and NSP4, and wherein R₁ R₂, R₄, and R₅ encode arotavirus protein that is not X₃, where X₄ may be any rotavirus proteinselected from the group of VP2, VP4, VP6, VP7 and NSP4, and whereinR₁-R₃ and R₅ encode a rotavirus protein that is not X₄, where X₅ may beany rotavirus protein selected from the group of VP2, VP4, VP6, VP7 andNSP4, and wherein R₁-R₄ encode a rotavirus protein that is not X₅, withthe result that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 isexpressed in the host.

For example which is not to be considered limiting, the first nucleicacid (N₁) may comprise a nucleotide sequence R₁, wherein R₁ may encoderotavirus protein VP2, VP4, VP6, VP7 or NSP4 and a second nucleotidesequence R₂ which may encode any rotavirus protein selected from thegroup of VP2, VP4, VP6, VP7 and NSP4 that is not encoded by R₁. Thesecond nucleotide sequence may comprise nucleotide sequences R₃ and R₄,wherein R₃ encode rotavirus protein VP2, VP4, VP6, VP7 or NSP4, but nota rotavirus protein that are encoded by R₁ or R₂ and R₄ encodesrotavirus protein VP2, VP4, VP6, VP7 or NSP4, but not a rotavirusprotein that are encoded by R₁, R₂ or R₃. The third nucleotide sequencemay comprise nucleotide sequences R₅, wherein R₅ encode rotavirusprotein VP2, VP4, VP6, VP7 or NSP4, but not a rotavirus protein that areencoded by R₁, R₂, R₃ or R₄, and wherein VP2, VP4, VP6, VP7 or NSP4 areencoded once. For example nucleotide sequence R₁ may encode rotavirusprotein VP2 and nucleotide sequence R₂-R₅ may encode in any orderrotavirus protein VP4, VP6, VP7 and NSP4, but R₂-R₅ may not encode VP2.In another example nucleotide sequence R₁ may encode rotavirus proteinVP4 and nucleotide sequences R₂-R₅ may encode in any order rotavirusprotein VP2, VP6, VP7 and NSP4. In yet another example nucleotidesequence R₁ may encode rotavirus protein VP6 and nucleotide sequencesR₂-R₅ may encode in any order rotavirus protein VP2, VP4, VP7 and NSP4.In yet another example nucleotide sequence R₁ may encode rotavirusprotein VP7 and nucleotide sequences R₂-R₅ may encode in any orderrotavirus protein VP2, VP4, VP6 and NSP4. In yet another examplenucleotide sequence R₁ may encode rotavirus protein NSP4 and nucleotidesequences R₂-R₅ may encode in any order rotavirus protein VP2, VP4, VP6and VP7.

A plant that expresses a first nucleic acid (N₁) comprising a firstnucleotide sequence (R₁) encoding a first rotavirus protein (X₁) may betransformed with a second nucleic acid (N₂) comprising a secondnucleotide sequence (R₂) encoding a second rotavirus protein (X₂). Theplant may be further transformed with a third nucleic acid (N₃)comprising a third nucleotide sequence (R₃) encoding a third rotavirusprotein (X₃), a fourth nucleotide sequences (R₄) encoding a fourthrotavirus protein (X₄) and a fifth nucleotide sequence (R₅) encoding afifth rotavirus protein (X₅), so that the first, second, third, fourthand fifth nucleotide sequence are co-expressed in the plant. Rotavirusprotein X₁-X₅ may be selected from the group of VP2, VP4, VP6, VP7 andNSP4, wherein VP2, VP4, VP6, VP7 and NSP4 are selected once, so thateach rotavirus protein VP2, VP4, VP6, VP7 and NSP4 is expressed in theplant. Furthermore, a plant may be simultaneously co-transformed with afirst nucleic acid (N₁) comprising a first nucleotide sequence (R₁)encoding a first rotavirus protein (X₁), a second nucleic acid (N₂)comprising a second nucleotide sequence (R₂) encoding a second rotavirusprotein (X₂), and a third nucleic acid (N₃) comprising a third, a fourthand a five nucleotide sequences (R₃-R₅) encoding a third, a fourth and afifth rotavirus protein (X₃-X₅), so that the first, second, third,fourth and fifth nucleotide sequence are co-expressed in the plant. Thefirst nucleic acid (N₁), second nucleic acid (N₂) and third nucleic acid(N₃) may be introduced in the plant in a transient manner, or in astable manner.

For example, a first plant expressing a first nucleic acid (N₁)comprising a first, second and third nucleotide sequence (R₁, R₂, andR₃) encoding a first, second and third rotavirus protein (X₁, X₂, andX₃) may be crossed with a second plant expressing a second nucleic acid(N₂) comprising a fourth nucleotide sequence (R₄) encoding a fourthrotavirus protein (X₄) to produce a progeny plant (third plant). Thethird plant may be crossed with a fourth plant expressing a thirdnucleic acid (N₃) comprising a fifth nucleotide sequence (R₅) encoding afifth rotavirus protein (X₅) to produces a progeny plant thatco-expresses the first, second, third, fourth and fifth rotavirusprotein (X₁-X₅). Furthermore, a first plant expressing a first nucleicacid (N₁) comprising a first nucleotide sequence (R₁) encoding a firstrotavirus protein (X₁) may be crossed with a second plant expressing asecond nucleic acid (N₂) comprising a second nucleotide sequence (R₂)encoding a rotavirus protein (X₂) to produce a progeny plan (thirdplant). The third plant may be crossed with a fourth plant expressing athird nucleic acid (N₃) comprising a third, fourth and fifth nucleotidesequence (R₃, R₄ and R₅) encoding a third, fourth and fifth rotavirusprotein (X₃, X₄, and X₅) to produce a progeny plant that co-expressesthe first, second, third, fourth and fifth rotavirus protein (X₁-X₅).Rotavirus protein X₁-X₅ may be selected from the group of VP2, VP4, VP6,VP7 and NSP4, so that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4is expressed in the plant.

3.3 Two Single Gene Constructs+One Dual Gene Construct

An alternated method of producing RLPs in a plant is also provided,wherein a first nucleic acid (N₁) comprising a nucleotide sequence (R₁)encoding a first rotavirus protein (X₁) is co-expressed with a secondnucleic acid (N₂) comprising a second nucleotide sequence (R₂) encodinga second rotavirus protein (X₂) and a third nucleic acid (N₃) comprisinga third nucleotide sequence (R₃) encoding a third rotavirus protein(X₃), and a fourth nucleotide sequence (R₄) encoding a fourth rotavirusprotein (X₄), so that the first, second, third and fourth nucleotidesequence are co-expressed in the plant (See Table 1, Combination #3.3).

As a non-limiting example, N₁ comprises (R₁), N₂ comprises (R₂) and N₃comprises (R₃, R₄), wherein each rotavirus protein selected from thegroup of VP2, VP4, VP6, and NSP4 is encoded once and wherein R₁ encodesrotavirus protein X₁, wherein X₁ may be any rotavirus protein selectedfrom the group of VP2, VP4, VP6, and NSP4, and wherein R₂-R₄ encode arotavirus protein that is not X₁, where X₂ may be any rotavirus proteinselected from the group of VP2, VP4, VP6, and NSP4, and wherein R₁ andR₃-R₄ encode a rotavirus protein that is not X₂, where X₃ may be anyrotavirus protein selected from the group of VP2, VP4, VP6, and NSP4,and wherein R₁ R₂, and R₄ encode a rotavirus protein that is not X₃,where X₄ may be any rotavirus protein selected from the group of VP2,VP4, VP6, and NSP4, and wherein R₁-R₃ encode a rotavirus protein that isnot X₄, with the result that each rotavirus protein VP2, VP4, VP6, andNSP4 is expressed in the host.

As another non-limiting example, N₁ comprises (R₁), N₂ comprises (R₂)and N₃ comprises (R₃, R₄), wherein each rotavirus protein selected fromthe group of VP2, VP7, VP6, and NSP4 is encoded once and wherein R₁encodes rotavirus protein X₁, wherein X₁ may be any rotavirus proteinselected from the group of VP2, VP7, VP6, and NSP4, and wherein R₂-R₄encode a rotavirus protein that is not X₁, where X₂ may be any rotavirusprotein selected from the group of VP2, VP7, VP6, and NSP4, and whereinR₁ and R₃-R₄ encode a rotavirus protein that is not X₂, where X₃ may beany rotavirus protein selected from the group of VP2, VP7, VP6, andNSP4, and wherein R₁ R₂, and R₄ encode a rotavirus protein that is notX₃, where X₄ may be any rotavirus protein selected from the group ofVP2, VP7, VP6, and NSP4, and wherein R₁-R₃ encode a rotavirus proteinthat is not X₄, with the result that each rotavirus protein VP2, VP6,VP7 and NSP4 is expressed in the host.

As a further non-limiting example, N₁ comprises (R₁), N₂ comprises (R₂)and N₃ comprises (R₃, R₄), wherein each rotavirus protein selected fromthe group of VP4, VP7, VP6, and NSP4 is encoded once and wherein R₁encodes rotavirus protein X₁, wherein X₁ may be any rotavirus proteinselected from the group of VP4, VP7, VP6, and NSP4, and wherein R₂-R₄encode a rotavirus protein that is not X₁, where X₂ may be any rotavirusprotein selected from the group of VP4, VP7, VP6, and NSP4, and whereinR₁ and R₃-R₄ encode a rotavirus protein that is not X₂, where X₃ may beany rotavirus protein selected from the group of VP4, VP7, VP6, andNSP4, and wherein R₁ R₂, and R₄ encode a rotavirus protein that is notX₃, where X₄ may be any rotavirus protein selected from the group ofVP4, VP7, VP6, and NSP4, and wherein R₁-R₃ encode a rotavirus proteinthat is not X₄, with the result that each rotavirus protein VP4, VP7,VP6, and NSP4 is expressed in the host.

3.4 Three Single Gene Constructs

An alternated method of producing RLPs in a plant is also provided,wherein a first nucleic acid (N₁) comprising a nucleotide sequence (R₁)encoding a first rotavirus protein (X₁) is co-expressed with a secondnucleic acid (N₂) comprising a second nucleotide sequence (R₂) encodinga second rotavirus protein (X₂) and a third nucleic acid (N₃) comprisinga third nucleotide sequence (R₃) encoding a third rotavirus protein(X₃), so that the first, second and third nucleotide sequence areco-expressed in the plant (See Table 1, Combination #3.4).

As a non-limiting example, N₁ comprises (R₁), N₂ comprises (R₂) and N₃comprises (R₃), wherein each rotavirus protein selected from the groupof VP4, VP6, and NSP4 is encoded once and wherein R₁ encodes rotavirusprotein X₁, wherein X₁ may be any rotavirus protein selected from thegroup of VP4, VP6, and NSP4, and wherein R₂-R₃ encode a rotavirusprotein that is not X₁, where X₂ may be any rotavirus protein selectedfrom the group of VP4, VP6, and NSP4, and wherein R₁ and R₃ encode arotavirus protein that is not X₂, where X₃ may be any rotavirus proteinselected from the group of VP4, VP6, and NSP4, and wherein R₁ and R₂encode a rotavirus protein that is not X₃, with the result that eachrotavirus protein VP4, VP6, and NSP4 is expressed in the host.

As a further non-limiting example, N₁ comprises (R₁), N₂ comprises (R₂)and N₃ comprises (R₃), wherein each rotavirus protein selected from thegroup of VP7, VP6, and NSP4 is encoded once and wherein R₁ encodesrotavirus protein X₁, wherein X₁ may be any rotavirus protein selectedfrom the group of VP7, VP6, and NSP4, and wherein R₂-R₃ encode arotavirus protein that is not X₁, where X₂ may be any rotavirus proteinselected from the group of VP7, VP6, and NSP4, and wherein R₁ and R₃encode a rotavirus protein that is not X₂, where X₃ may be any rotavirusprotein selected from the group of VP7, VP6, and NSP4, and wherein R₁and R₂ encode a rotavirus protein that is not X₃, with the result thateach rotavirus protein VP7, VP6, and NSP4 is expressed in the host.

4. Four Constructs

4.1. Three Single Gene Constructs+One Dual Gene Construct

Also provided herein is a method of producing RLPs in a plant, wherein afirst nucleic acid (N₁) comprising a first nucleotide sequence (R₁)encoding a first rotavirus protein (X₁) is co-expressed with a secondnucleic acid (N₂) comprising a second nucleotide sequence (R₂) encodinga second rotavirus protein (X₂), a third nucleic acid (N₃) comprising athird nucleotide sequence (R₃) encoding a third rotavirus protein (X₃),and fourth nucleic acid (N₄) comprising a fourth nucleotide sequences(R₄) encoding a fourth rotavirus protein (X₄) and a fifth nucleotidesequence (R₅) encoding a fifth rotavirus protein (X₅) (see Table 1,Combination #4.1) so that the first, second, third, fourth and fifthnucleotide sequence are co-expressed in the plant.

In this example, N₁ comprises (R₁), N₂ comprises (R₂), N₃ comprises(R₃), and N₄ comprises (R₄, and R₅), wherein each rotavirus proteinselected from the group of VP2, VP4, VP6, VP7 and NSP4 is encoded, andwherein R₁ encodes rotavirus protein X₁, wherein X₁ may be any rotavirusprotein selected from the group of VP2, VP4, VP6, VP7 and NSP4, andwherein R₂-R₅ encode a rotavirus protein that is not X₁, where X₂ may beany rotavirus protein selected from the group of VP2, VP4, VP6, VP7 andNSP4, and wherein R₁ and R₃-R₅ encode a rotavirus protein that is notX₂, where X₃ may be any rotavirus protein selected from the group ofVP2, VP4, VP6, VP7 and NSP4, and wherein R₁ R₂, R₄, and R₅ encode arotavirus protein that is not X₃, where X₄ may be any rotavirus proteinselected from the group of VP2, VP4, VP6, VP7 and NSP4, and whereinR₁-R₃ and R₅ encode a rotavirus protein that is not X₄, where X₅ may beany rotavirus protein selected from the group of VP2, VP4, VP6, VP7 andNSP4, and wherein R₁-R₄ encode a rotavirus protein that is not X₅, withthe result that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 isexpressed in the host.

The four nucleic acids may be introduced into a plant in any order. Forexample, which is not be considered limiting, a plant that expresses afirst nucleic acid (N₁) comprising a first nucleotide sequence (R₁)encoding a first rotavirus protein (X₁) may be transformed with a secondnucleic acid (N₂) comprising a second nucleotide sequence (R₂) encodinga second rotavirus protein (X₂). The plant may be further transformedwith a third nucleic acid (N₃) comprising a third nucleotide sequence(R₃) encoding a third rotavirus protein (X₃). The plant then may befurther be transformed with a fourth nucleic acid (N₄) comprising afourth nucleotide sequences (R₄) encoding a fourth rotavirus protein(X₄) and a fifth nucleotide sequence (R₅) encoding a fifth rotavirusprotein (X₅), so that the first, second, third, fourth and fifthnucleotide sequence are co-expressed in the plant. Furthermore, a plantmay be simultaneously co-transformed with a first nucleic acid (N₁)comprising a first nucleotide sequence (R₁) encoding a first rotavirusprotein (X₁), a second nucleic acid (N₂) comprising a second nucleotidesequence (R₂) encoding a second rotavirus protein (X₂), a third nucleicacid (N₃) comprising a third nucleotide sequence (R₃) encoding a thirdrotavirus protein (X₃), and a fourth nucleic acid (N₄) comprising afourth and a fifth nucleotide sequences (R₄-R₅) encoding a fourth and afifth rotavirus protein (X₄-X₅) so that the first, second, third, fourthand fifth nucleotide sequence are co-expressed in the plant. The firstnucleic acid (N₁), second nucleic acid (N₂), third nucleic acid (N₃) andfourth nucleic acid (N₄) may be introduced in the plant in a transientmanner, or in a stable manner.

Furthermore, a plant that expresses a first nucleic acid (N₄) comprisinga first nucleotide sequence (R₄) encoding a first rotavirus protein (X₄)and a second nucleotide sequence (R₅) encoding a second rotavirusprotein (X₅) may be transformed with a second nucleic acid (N₁)comprising a third nucleotide sequence (R₁) encoding a third rotavirusprotein (X₁). The plant may be further transformed with a third nucleicacid (N₂) comprising a third nucleotide sequence (R₂) encoding a fourthrotavirus protein (X₃). The plant then may be further be transformedwith a fourth nucleic acid (N₃) comprising a fifth nucleotide sequences(R₃) encoding a fifth rotavirus protein and a fifth nucleotide sequence(R₃) encoding a fifth rotavirus protein (X₃), so that the first, second,third, fourth and fifth nucleotide sequence are co-expressed in theplant. Rotavirus protein X₁-X₅ may be selected from the group of VP2,VP4, VP6, VP7 and NSP4, wherein VP2, VP4, VP6, VP7 and NSP4 are selectedonce, so that each rotavirus protein VP2, VP4, VP6, VP7 and NSP4 isexpressed in the plant. The first nucleic acid (N₄), second nucleic acid(N₁), third nucleic acid (N₂) and fourth nucleic acid (N₃) may beintroduced in the plant in a transient manner, or in a stable manner.

A first plant expressing a first nucleic acid (N₁) comprising a firstnucleotide sequence (R₁) encoding a first rotavirus protein (X₁), may becrossed with a second plant expressing a second nucleic acid (N₂)comprising a second nucleotide sequence (R₂) encoding a second rotavirusprotein (X₂) to produce a progeny plant (third plant).

The third plant may be crossed with a fourth plant expressing a thirdnucleic acid (N₃) comprising a third nucleotide sequence (R₃) encoding athird rotavirus protein (X₃) to produces a progeny plant (fifth plant).The fifth plant may be crossed with a sixth plant expressing a fourthnucleic acid (N₄) comprising a fourth nucleotide sequences (R₄) encodinga fourth rotavirus protein (X₄) and a fifth nucleotide sequences (R₅)encoding a fifth rotavirus protein (X₅) to produce a progeny plant thatco-expresses the first, second, third, fourth and fifth rotavirusprotein (X₁-X₅).

Furthermore, a first plant expressing a first nucleic acid (N₄)comprising a first nucleotide sequence (R₄) encoding a first rotavirusprotein (X₄) and a second nucleotide sequence (R₅) encoding a secondrotavirus protein (X₅) may be crossed with a second plant expressing asecond nucleic acid (N₂) comprising a third nucleotide sequence (R₁)encoding a third rotavirus protein (X₁) to produce a progeny plan (thirdplant). The third plant may be crossed with a fourth plant expressing athird nucleic acid (N₂) comprising a fourth nucleotide sequence (R₂)encoding a fourth rotavirus protein (X₂) to produce a progeny plant(fifth plant). The fifth plant may be crossed with a sixth plantexpressing a fifth nucleotide sequence (R₃) encoding a fifth rotavirusprotein (X₃) to produce a progeny plant that co-expresses the first,second, third, fourth and fifth rotavirus protein (X₁-X₅). Rotavirusprotein X₁-X₅ may be selected from the group of VP2, VP4, VP6, VP7 andNSP4, wherein VP2, VP4, VP6, VP7 and NSP4 are selected once, so thateach rotavirus protein VP2, VP4, VP6, VP7 and NSP4 is expressed in theplant.

4.2 Four Single Gene Constructs

Also provided herein is a method of producing RLPs in a plant, wherein afirst nucleic acid (N₁) comprising a first nucleotide sequence (R₁)encoding a first rotavirus protein (X₁) is co-expressed with a secondnucleic acid (N₂) comprising a second nucleotide sequence (R₂) encodinga second rotavirus protein (X₂), a third nucleic acid (N₃) comprising athird nucleotide sequence (R₃) encoding a third rotavirus protein (X₃),and a fourth nucleic acid (N₄) comprising a fourth nucleotide sequences(R₄) encoding a fourth rotavirus protein (X₄), so that the first,second, third, fourth and fifth nucleotide sequence are co-expressed inthe plant (See Table 1, Combination #4.2).

In a non-limiting example, N₁ comprises (R₁), N₂ comprises (R₂), N₃comprises (R₃), and N₄ comprises (R₄), wherein each rotavirus proteinselected from the group of VP2, VP4, VP6 and NSP4 is encoded, andwherein R₁ encodes rotavirus protein X₁, wherein X₁ may be any rotavirusprotein selected from the group of VP2, VP4, VP6 and NSP4, and whereinR₂-R₄ encode a rotavirus protein that is not X₁, where X₂ may be anyrotavirus protein selected from the group of VP2, VP4, VP6 and NSP4, andwherein R₁ and R₃-R₄ encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP2, VP4, VP6and NSP4, and wherein R₁ R₂, and R₄ encode a rotavirus protein that isnot X₃, where X₄ may be any rotavirus protein selected from the group ofVP2, VP4, VP6 and NSP4, and wherein R₁-R₃ encode a rotavirus proteinthat is not X₄, with the result that each rotavirus protein VP2, VP4,VP6 and NSP4 is expressed in the host.

In another non-limiting example, N₁ comprises (R₁), N₂ comprises (R₂),N₃ comprises (R₃), and N₄ comprises (R₄), wherein each rotavirus proteinselected from the group of VP2, VP7, VP6 and NSP4 is encoded, andwherein R₁ encodes rotavirus protein X₁, wherein X₁ may be any rotavirusprotein selected from the group of VP2, VP7, VP6 and NSP4, and whereinR₂-R₄ encode a rotavirus protein that is not X₁, where X₂ may be anyrotavirus protein selected from the group of VP2, VP7, VP6 and NSP4, andwherein R₁ and R₃-R₄ encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP2, VP7, VP6and NSP4, and wherein R₁ R₂, and R₄ encode a rotavirus protein that isnot X₃, where X₄ may be any rotavirus protein selected from the group ofVP2, VP7, VP6 and NSP4, and wherein R₁-R₃ encode a rotavirus proteinthat is not X₄, with the result that each rotavirus protein VP2, VP7,VP6 and NSP4 is expressed in the host.

In a further non-limiting example, N₁ comprises (R₁), N₂ comprises (R₂),N₃ comprises (R₃), and N₄ comprises (R₄), wherein each rotavirus proteinselected from the group of VP4, VP7, VP6 and NSP4 is encoded, andwherein R₁ encodes rotavirus protein X₁, wherein X₁ may be any rotavirusprotein selected from the group of VP4, VP7, VP6 and NSP4, and whereinR₂-R₄ encode a rotavirus protein that is not X₁, where X₂ may be anyrotavirus protein selected from the group of VP4, VP7, VP6 and NSP4, andwherein R₁ and R₃-R₄ encode a rotavirus protein that is not X₂, where X₃may be any rotavirus protein selected from the group of VP4, VP7, VP6and NSP4, and wherein R₁ R₂, and R₄ encode a rotavirus protein that isnot X₃, where X₄ may be any rotavirus protein selected from the group ofVP4, VP7, VP6 and NSP4, and wherein R₁-R₃ encode a rotavirus proteinthat is not X₄, with the result that each rotavirus protein VP4, VP7,VP6 and NSP4 is expressed in the host.

5. Five Constructs

5. Five Single Gene Constructs

The present invention also provides for a method of producing RLPs in aplant, wherein a first nucleic acid (N₁) comprising a first nucleotidesequence (R₁) encoding a first rotavirus protein (X₁) is co-expressedwith a second nucleic acid (N₂) comprising a second nucleotide sequence(R₂) encoding a second rotavirus protein (X₂) and a third nucleic acid(N₃) comprising a third nucleotide sequence (R₃) encoding a thirdrotavirus protein (X₃) and fourth nucleic acid (N₄) comprising a fourthnucleotide sequences (R₄) encoding a fourth rotavirus protein (X₄) and afifth nucleic acid (R₅) comprising a fifth nucleotide sequence (R₅)encoding a fifth rotavirus protein (X₅) (see Table 1, Combination #5) sothat the first, second, third, fourth and fifth nucleotide sequence areco-expressed in the plant.

In this non-limiting example, N₁ comprises (R₁), N₂ comprises (R₂), N₃comprises (R₃), N₄ comprises (R₄) and N₅ comprises (R₅), wherein eachrotavirus protein selected from the group of VP2, VP4, VP6, VP7 andNSP4, and wherein R₁ encodes rotavirus protein X₁, wherein X₁ may be anyrotavirus protein selected from the group of VP2, VP4, VP6, VP7 andNSP4, and wherein R₂-R₅ encode a rotavirus protein that is not X₁, whereX₂ may be any rotavirus protein selected from the group of VP2, VP4,VP6, VP7 and NSP4, and wherein R₁ and R₃-R₅ encode a rotavirus proteinthat is not X₂, where X₃ may be any rotavirus protein selected from thegroup of VP2, VP4, VP6, VP7 and NSP4, and wherein R₁ R₂, R₄, and R₅encode a rotavirus protein that is not X₃, where X₄ may be any rotavirusprotein selected from the group of VP2, VP4, VP6, VP7 and NSP4, andwherein R₁-R₃ and R₅ encode a rotavirus protein that is not X₄, where X₅may be any rotavirus protein selected from the group of VP2, VP4, VP6,VP7 and NSP4, and wherein R₁-R₄ encode a rotavirus protein that is notX₅, with the result that each rotavirus protein VP2, VP4, VP6, VP7 andNSP4 is expressed in the host.

For example, which is not to be considered limiting, nucleotide sequenceR₁ may encode rotavirus protein VP2 and nucleotide sequence R₂-R₅ mayencode in any order rotavirus protein VP4, VP6, VP7 and NSP4, but R₂-R₅may not encode VP2. In another non-limiting example nucleotide sequenceR₁ may encode rotavirus protein VP4 and nucleotide sequences R₂-R₅ mayencode in any order rotavirus protein VP2, VP6, VP7 and NSP4. In yetanother non-limiting example nucleotide sequence R₁ may encode rotavirusprotein VP6 and nucleotide sequences R₂-R₅ may encode in any orderrotavirus protein VP2, VP4, VP7 and NSP4. In yet another example whichis not to be considered limiting, nucleotide sequence R₁ may encoderotavirus protein VP7 and nucleotide sequences R₂-R₅ may encode in anyorder rotavirus protein VP2, VP4, VP6 and NSP4. In yet anothernon-limiting example nucleotide sequence R₁ may encode rotavirus proteinNSP4 and nucleotide sequences R₂-R₅ may encode in any order rotavirusprotein VP2, VP4, VP6 and VP7.

For example, which is not to be considered limiting, a first nucleicacid (N₁) comprising a first nucleotide sequence (R₁) encoding a firstrotavirus protein, for example VP2 is co-expressed with a second nucleicacid (N₂) comprising a second nucleotide sequence (R₂) encoding a secondrotavirus protein, for example VP6, a third nucleic acid (N₃) comprisinga third nucleotide sequence (R₃) encoding a third rotavirus protein forexample VP4, a fourth nucleic acid (N₄) comprising a fourth nucleotidesequence (R₄) encoding a fourth rotavirus protein, for example VP7 and afifth nucleic acid (N5) comprising a fifth nucleotide sequence (R₅)encoding a fifth rotavirus protein for example NSP4 (see FIG. 3).

The five nucleic acids may be introduced into a plant in any order. Forexample, which is not to be considered limiting, a plant that expressesa first nucleic acid (N₁) comprising a first nucleotide sequence (R₁)encoding a first rotavirus protein (X₁) may be transformed with a secondnucleic acid (N₂) comprising a second nucleotide sequence (R₂) encodinga second rotavirus protein (X₂). The plant may be further transformedwith a third nucleic acid (N₃) comprising a third nucleotide sequence(R₃) encoding a third rotavirus protein (X₃). The plant then may befurther transformed with a fourth nucleic acid (N₄) comprising a fourthnucleotide sequences (R₄) encoding a fourth rotavirus protein (X₄). Theplant then may be further transformed with a fifth nucleic acid (N₅)comprising a fourth nucleotide sequences (R₅) encoding a fourthrotavirus protein (X₅), so that the first, second, third, fourth andfifth nucleotide sequence are co-expressed in the plant. Rotavirusprotein X₁-X₅ may be selected from the group of VP2, VP4, VP6, VP7 andNSP4, wherein VP2, VP4, VP6, VP7 and NSP4 are selected once, so thateach rotavirus protein VP2, VP4, VP6, VP7 and NSP4 is expressed in theplant. Furthermore, a plant may be co-transformed simultaneously with afirst nucleic acid (N₁) comprising a first nucleotide sequence (R₁)encoding a first rotavirus protein (X₁), a second nucleic acid (N₂)comprising a second nucleotide sequence (R₂) encoding a second rotavirusprotein (X₂), a third nucleic acid (N₃) comprising a third nucleotidesequence (R₃) encoding a third rotavirus protein (X₃), a fourth nucleicacid (N₄) comprising a fourth nucleotide sequences (R₄) encoding afourth and a fifth rotavirus protein (X₄), and a fifth nucleic acid (N₅)comprising a fifth nucleotide sequences (R₅) encoding a fifth rotavirusprotein (X₅) so that the first, second, third, fourth and fifthnucleotide sequence are co-expressed in the plant. The first nucleicacid (N₁), second nucleic acid (N₂), third nucleic acid (N₃), fourthnucleic acid (N₄) and fifth nucleic acid (N₅) may be introduced in theplant in a transient manner, or in a stable manner.

A first plant expressing a first nucleic acid (N₁) comprising a firstnucleotide sequence (R₁) encoding a first rotavirus protein (X₁), may becrossed with a second plant expressing a second nucleic acid (N₂)comprising a second nucleotide sequence (R₂) encoding a second rotavirusprotein (X₂) to produce a progeny plant (third plant). The third plantmay be crossed with a fourth plant expressing a third nucleic acid (N₃)comprising a third nucleotide sequence (R₃) encoding a third rotavirusprotein (X₃) to produces a progeny plant (fifth plant). The fifth plantmay be crossed with a sixth plant expressing a fourth nucleic acid (N₄)comprising a fourth nucleotide sequences (R₄) encoding a fourthrotavirus protein (X₄) to produce a progeny plant (seventh plant). Theseventh plant may be crossed with an eight plant expressing a fifthnucleic acid (N₅) comprising a fifth nucleotide sequences (R₅) encodinga fifth rotavirus protein (X₅) to produce a progeny plant thatco-expresses the first, second, third, fourth and fifth rotavirusprotein (X₁-X₅). Rotavirus protein X₁-X₅ may be selected from the groupof VP2, VP4, VP6, VP7 and NSP4, wherein VP2, VP4, VP6, VP7 and NSP4 areselected once, so that each rotavirus protein VP2, VP4, VP6, VP7 andNSP4 is expressed in the plant.

Ratio of Nucleic Acids (N) used to Transform a Host

As may be seen in FIG. 5, the level of RLP accumulation in the plant,portion of the plant or plant cell, may be influenced by the ratio ofthe nucleic acids encoding rotavirus structural and nonstructuralproteins that are expressed in a plant. For example, which is not to beconsidered limiting, the ratio of nucleic acids (N) that are introducedinto a plant may be modified by providing different amounts ofAgrobacterium, that are used to infiltrate the plant, portion of theplant or plant cell, where each Agrobacterium comprises a constructcomprising a nucleic acid (N) as set out in Table 1 (and accompanyingtext) above. For example which is not to be considered limiting, theratio of the structural protein-containing to nonstructuralprotein-containing Agrobacterium may range for example from about 0.8:1to about 2.5:1.5 (structural protein:nonstructural protein), or anyamount therebetween, for example from about 0.8:1, 0.9:1, 1:1, 1.1:1,1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:0.5, 2:1,2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.5:1.1, 2.5:1.2, 2.5:1.3, 2.5:1.4, 2.5:1.5(structural protein:nonstructural protein), or any amount therebetween.For example, as described below, the ratio of structural rotavirusprotein to nonstructural protein may be varied for example byintroducing different ratios of Agrobacterium containing the one or morenucleic acid (N) comprising one or more nucleotide sequence (R) encodingthe rotavirus structural and non-structural proteins (X) into a host,for example a plant, portion of the plant or plant cell. For example theabsorbance or optical density (OD) of Agrobacterium strains in thebacterial suspension may be used as measure to establish a ratio betweenAgrobacterium strains that containing the structural protein toAgrobacterium strains that contain the nonstructural protein. Forexample, which is not considered limiting, the OD may range for examplefrom about 0.2:0.4 to about 1:0.6 (Agrobacterium strains containingstructural protein:Agrobacterium strains containing nonstructuralprotein in the bacterial suspension) or any amount therebetween, forexample from about 0.2:0.4, 0.3:0.4, 0.4:0.4, 0.5:0.4, 0.6:0.4, 0.7:0.4,0.8:0.4, 0.9:0.4, 1:0.4, 0.2:0.5, 0.3:0.5, 0.4:0.5, 0.5:0.5, 0.6:0.5,0.7:0.5, 0.8:0.5, 0.9:0.5, 1:05, 0.2:0.6, 0.3:0.6, 0.4:0.6, 0.5:0.6,0.6:0.6, 0.7:0.6, 0.8:0.6, 0.9:0.6, 1:06, 0.3:0.4, 0.3:0.5, 0.3:0.6,0.4:0.4, 0.4:0.5, 0.4:0.6, 0.5:0.4, 0.5:0.5, 0.5:0.6, 0.6:0.4, 0.6:0.5,0.6:0.6, 0.7:0.4, 0.7:0.5, 0.7:0.6, 0.8:0.4, 0.8:0.5, 0.8:0.6, 0.9:0.4,0.9:0.5, 0.9:0.6, 1:0.4, 1:0.5, 1:0.6 (Agrobacterium strains containingstructural protein:Agrobacterium strains containing nonstructuralprotein in the bacterial suspension) or any amount therebetween. Forexample, which is not considered limiting, an OD of 0.4 of Agrobacteriumstrains in bacterial suspension may be designated as a reference of 1.Therefore a ratio of 1.5:1 of structural to non structural protein maybe achieved by using an OD of 0.6 of Agrobacterium strain containingstructural protein to an OD of 0.4 of Agrobacterium strain containingnonstructural protein in bacterial suspension.

The ratio of rotavirus structural protein to nonstructural protein maybe varied for example by introducing different ratios of Agrobacteriumcontaining the one or more nucleic acid (N) comprising one or morenucleotide sequence (R) encoding the rotavirus structural andnon-structural proteins (X) into the plant, portion of the plant orplant cell. Alternatively, if the rotavirus structural proteins andnonstructural proteins are present on the same construct, and thereforeare introduced into the plant, plant portion or plant cell, using oneAgrobacterium, they may be differentially expressed within the plant,portion of the plant or plant cell using suitable promoters so that thedesired ratio of rotavirus structural proteins and nonstructuralproteins is obtained.

Therefore as described herein, a method is provided for increasing RLPproduction yield, increasing VP4 and VP7 yield, or increasing both RLPSand VP4 and VP7 yield, by modulating the ratio between the one or morenucleic acid (N) comprising one or more nucleotide sequence (R) encodingthe rotavirus structural proteins (X) and the one or more nucleic acid(N) comprising one or more nucleotide sequence (R) encoding therotavirus nonstructural proteins (X).

For example, the percentage of the Agrobacterium containing rotavirusnonstructural protein may be between 20% to 60% or any amounttherebetween, of total amount of Agrobacterium use to infiltrate theplant, plant portion or plant cell. For example the percent ratio ofAgrobacterium containing rotavirus nonstructural protein may be 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,49% 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, or any amounttherebetween of the total Agrobacterium use to infiltrate the plant,plant portion or plant cell. Similarly, the percentage of Agrobacteriumcontaining structural protein within the total amount of Agrobacteriuminfiltrated may be 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%,70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%,56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%,42%, 41% or 40% or any amount therebetween, of the total Agrobacteriumuse to infiltrate the plant, plant portion or plant cell.

For example, the percentage ratio of Agrobacterium containing rotavirusstructural protein to Agrobacterium containing nonstructural protein maybe 70%:30%, 60%:40%, 50%:50%, 40%:60% or any percentage ratio amounttherebetween. For example, the percentage ratio between Agrobacteriumcontaining structural protein and Agrobacterium containing nonstructuralprotein may be 50%:50%, 51%:49%, 52%:48%, 53%:47%, 54%:46%, 55%:45%,56%:44%, 57%:43%, 58%:42%, 59%: 41%, 60%:40%, or any percentage ratio inbetween.

As described below, the ratio of rotavirus structural protein torotavirus nonstructural protein may further be varied for example bydifferentially expressing the rotavirus structural protein and therotavirus nonstructural protein. Expression may be varied by modulatingfor example replication, transcription, translation, or a combinationthereof, of the rotavirus structural protein, the rotavirusnonstructural protein, or both the rotavirus structural protein and therotavirus nonstructural protein. For example different regulatoryelements, including promoters, amplification elements, enhancers or acombination thereof, may be used in addition to varying the ratio of therotavirus structural protein-containing Agrobacterium to rotavirusnonstructural protein-containing Agrobacterium infiltrated as describedabove. A first set or combination of regulatory elements may be used toregulate the replication, transcription or a combination thereof, of theone or more nucleic acid comprising one or more nucleotide sequenceencoding rotavirus structural protein and a second set or combination ofregulatory elements may be used to regulate the replication,transcription or a combination thereof, of the one or more nucleotidesequence encoding rotavirus nonstructural protein. The first set orcombination of regulatory elements is different from the second set orcombination of regulatory elements and permits differential expressionof the one or more nucleic acid comprising one or more nucleotidesequence encoding rotavirus structural protein and the one or morenucleic acid comprising one or more nucleotide sequence encodingrotavirus nonstructural protein to permit modulating the ratio ofrotavirus structural protein:rotavirus nonstructural protein in vivo.

For example, which is not to be considered limiting, one set orcombination of regulatory elements, for example the first set, mayinclude an enhancer element for example elements obtained from CPMV,such as CPMV HT, or CPMV 160 (see FIG. 6). CMPV HT is described in U.S.61/971,274 (which is incorporated herein by reference) and CPMV 160 isdescribed in U.S. 61/925,852 (which is incorporated herein byreference). The enhancer element, for example those obtained from CPMV,for example CPMV HT or CPMV 160 (see FIG. 6; U.S. 61/971,274, and U.S.61/925,852, respectively) may be absent in the other set or combinationof regulatory elements, for example the second set. Alternatively, thesecond set may include an enhancer element (for example elementsobtained from CPMV, (for example CPMV HT or CPMV 160), while theamplification element (for example elements obtained from CPMV, (forexample CPMV HT or CPMV 160) may be absent in the first set orcombination of regulatory elements. In a similar manner, the strength ofa promoters may differ between the first and second set or combinationof regulatory elements, or one of the promoters may be inducible, andthe other constitutive, so that differential expression between therotavirus structural protein relative to the rotavirus nonstructuralprotein is achieved in vivo.

For example, the ratio of rotavirus structural protein to nonstructuralprotein may be varied for example by introducing different ratios ofAgrobacterium containing a first nucleic acid (N₁) comprising anucleotide sequence (R₁) encoding first rotavirus protein for examplerotavirus nonstructural protein NSP4 to Agrobacterium containing asecond nucleic acid (N₂) comprising four nucleotide sequences (R₂-R₅)encoding a second, third, fourth and fifth rotavirus protein, forexample in any order rotavirus structural proteins VP2, VP4, VP6 andVP7. For example the ratio of the Agrobacterium containing a firstnucleic acid (N₁) comprising a nucleotide sequence (R₁) encodingrotavirus nonstructural protein NSP4 to the Agrobacterium containing asecond nucleic acid (N₂) comprising four nucleotide sequences (R₂-R₅)encoding rotavirus structural proteins VP2, VP4, VP6 and VP7 may be0.8:1 and 1:2 (Agrobacterium containing N₁ to N₂) or any amount therebetween for example 1:1.5 (Agrobacterium containing N₁ to N₂).

Furthermore, the ratio of rotavirus structural protein to nonstructuralprotein may be varied by differentially expressing within the plant,portion of the plant or plant cell the rotavirus structural protein tononstructural protein using enhancer elements. For example, the ratio ofrotavirus structural protein to nonstructural protein may be varied forexample by co-expressing within the plant, portion of the plant or plantcell a first nucleic acid (N₁) comprising a nucleotide sequence (R₁)encoding a first rotavirus protein for example a nonstructural proteinNSP4 with a second nucleic acid (N₂) comprising four nucleotidesequences (R₂-R₅) encoding a second, third, fourth and fifth rotavirusprotein for example in any order rotavirus structural proteins VP2, VP4,VP6 and VP7, wherein the second, third, fourth and fifth nucleotidesequence are operatively linked to an enhancer sequence for example CPMVHT, CPM 160, CPMV 160+ and CPMV HT+ (described in U.S. 61/971,274, andU.S. 61/925,852, respectively which are incorporated herein byreference), as described below. In another example, the ratio ofrotavirus structural protein to nonstructural protein may be varied forexample by co-expressing within the plant, portion of the plant or plantcell a first nucleic acid (N₁) comprising first nucleotide sequence (R₁)encoding a first rotavirus protein for example structural protein VP6 orVP7 and second nucleotide sequence (R₂) encoding a second rotavirusprotein for example structural protein VP2 or VP4, second nucleic acid(N₂) comprising a third nucleotide sequence (R₃) encoding a thirdrotavirus protein for example structural protein VP7 or VP6 and a fourthnucleotide sequence (R₄) encoding a fourth rotavirus protein for examplestructural protein VP4 or VP2 and a third nucleic acid (N₃) comprisingfifth nucleotide sequences (R₅) encoding a fifth rotavirus protein forexample nonstructural protein NSP4, wherein the first, second, third andfourth nucleotide sequence are operatively linked to an enhancersequence for example CPMV HT, CPMV 160, CPMV 160+ and CPMV HT+, asdescribed below.

In another example, the ratio of rotavirus structural protein tononstructural protein may be varied for example by co-expressing withinthe plant, portion of the plant or plant cell one or more nucleic acidscomprising a first nucleotide sequence (R₁) encoding a first rotavirusprotein for example structural protein VP6 or VP7 and second nucleotidesequence (R₂) encoding a second rotavirus protein for example structuralprotein VP2 or VP4, a third nucleotide sequence (R₃) encoding a thirdrotavirus protein for example structural protein VP7 or VP6 and a fourthnucleotide sequence (R₄) encoding fourth rotavirus protein for examplestructural protein VP4 or VP2, and a fifth nucleotide sequences (R₅)encoding a fifth rotavirus protein for example nonstructural proteinNSP4, wherein the first, second, third and fourth nucleotide sequenceare operatively linked to an enhancer sequence for example CPM 160, CPMV160+ and CPMV HT+, as described below and the fifth nucleotide sequenceis operatively linked to CPMV HT as described below.

In another example, the ratio of rotavirus structural protein tononstructural protein may be varied for example by co-expressing withinthe plant, portion of the plant or plant cell a first nucleic acid (N₁)comprising first nucleotide sequence (R₁) encoding a first rotavirusprotein for example structural protein VP6 or VP7, a second nucleic acid(N₂) comprising second nucleotide sequence (R₂) encoding a secondrotavirus protein for example structural protein VP2 or VP4, a thirdnucleic acid (N₃) comprising a third nucleotide sequence (R₃) encoding athird rotavirus protein for example structural protein VP7 or VP6, afourth nucleic acid (N₄) comprising a fourth nucleotide sequence (R₄)encoding a fourth rotavirus protein for example structural protein VP4or VP2 and a fifth nucleic acid (N₅) comprising fifth nucleotidesequences (R₅) encoding a fifth rotavirus protein for examplenonstructural protein NSP4, wherein the first, second, third and fourthnucleotide sequence are operatively linked to an enhancer sequence forexample CPMV HT, CPMV 160, CPMV 160+ and CPMV HT+, as described below.

Amplification Elements and Enhancer Elements/Regulatory Elements

The rotavirus protein or polypeptide may be expressed in an expressionsystem comprising a viral based, DNA or RNA, expression system, forexample but not limited to, a comovirus-based expression cassette andgeminivirus-based amplification element.

Enhancer elements may be used to achieve high level of transientexpression of rotavirus structural and nonstructural proteins. Enhancerelements may be based on RNA plant viruses, including comoviruses, suchas Cowpea mosaic virus (CPMV; see, for example, WO2007/135480;WO2009/087391; US 2010/0287670, Sainsbury F. et al., 2008, PlantPhysiology; 148: 121-1218; Sainsbury F. et al., 2008, PlantBiotechnology Journal; 6: 82-92; Sainsbury F. et al., 2009, PlantBiotechnology Journal; 7: 682-693; Sainsbury F. et al. 2009, Methods inMolecular Biology, Recombinant Proteins From Plants, vol. 483: 25-39).

CPMV 160 (CPMVX) and CPMV 160+ (CPMVX+)

In an embodiment the enhancer Elements are “CPMVX” (also referred as“CPMV 160”) and/or “CPMVX+” (also referred to as “CPMV 160+”) and aredescribed in U.S. 61/925,852 (which is incorporated herein byreference).

Expression enhancer “CPMVX” comprises a comovirus cowpea mosaic virus(CPMV) 5′ untranslated region (UTR). The 5′UTR from nucleotides 1-160 ofthe CPMV RNA-2 sequence (SEQ ID NO: 1), starts at the transcriptionstart site to the first in frame initiation start codon (at position161), which serve as the initiation site for the production of thelonger of two carboxy coterminal proteins encoded by a wild-typecomovirus genome segment. Furthermore a ‘third’ initiation site at (orcorresponding to) position 115 in the CPMV RNA-2 genomic sequence mayalso be mutated, deleted or otherwise altered. It has been shown thatremoval of AUG 115 in addition to the removal of AUG 161 enhancesexpression when combined with an incomplete M protein (Sainsbury andLomonossoff, 2008, Plant Physiology; 148: 1212-1218; WO 2009/087391;which are incorporated herein by reference).

CPMVX comprises X nucleotides of SEQ ID NO:1, where X=160, 155, 150, or114 of SEQ ID NO:1, or a sequence that comprises between 80% to 100%sequence similarity with CPMVX, where X=160, 155, 150, or 114 of SEQ IDNO:93. This expression enhancer is generally referred to as CPMVX (seeFIG. 6c ).

The expression enhancer CPMVX, where X=160, consists of nucleotides1-160 of SEQ ID NO: 1:

(SEQ ID NO: 1)   1tattaaaatc ttaataggtt ttgataaaag cgaacgtggg gaaacccgaa ccaaaccttc  61ttctaaactc tctctcatct ctcttaaagc aaacttctct cttgtctttc ttgcgtgagc 121gatcttcaac gttgtcagat cgtgcttcgg caccagtaca 

The CPMVX enhancer sequence may further be fused to a stuffer sequence,wherein the CMPVX comprises X nucleotides of SEQ ID NO:1, where X=160,155, 150, or 114 of SEQ ID NO:1, or a sequence that comprises between 80to 100% sequence similarity with CPMVX, where X=160, 155, 150, or 114 ofSEQ ID NO:1, and the stuffer sequence comprises from 1-100 nucleotidesfused to the 3′ end of the CMPVX sequence. For example, the stuffersequence may comprise from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides, or any number ofnucleotides therebetween.

If the CMPVX sequence comprises a stuffer fragment, then this expressionenhancer may be referred to as CPMVX+ (see FIG. 6d ), where X=160, 155,150, 114 of SEQ ID NO:1, it may also be referred to as CMPVX comprisinga stuffer sequence, or it may be referred to as CPMV160+; CPMV155+;CPMV150+; CPMV114+, when X-160, 155, 150, or 114, respectively.Constructs comprising CPMVX that do not comprise a stuffer sequence maybe termed CPMVX+, where X=160, 155, 150, 114 of SEQ ID NO:1, and wherethe stuffer sequence is of 0 nucleotides in length.

The stuffer sequence may be modified by truncation, deletion, orreplacement of the native CMPV 5′UTR sequence that is located 3′ tonucleotide 160. The modified stuffer sequence may be removed, replaced,truncated or shortened when compared to the initial or unmodified (i.e.native) stuffer sequence associated with the 5′UTR (as described inSainsbury F., and Lomonossoff G. P., 2008, Plant Physiol. 148: pp.1212-1218). The stuffer sequence may comprise a one or more restrictionsites (polylinker, multiple cloning site, one or more cloning sites),one or more plant kozak sequences, one or more linker sequences, one ormore recombination sites, or a combination thereof. For example, whichis not to be considered limiting, a stuffer sequence may comprise inseries, a multiple cloning site of a desired length fused to a plantkozak sequence. The stuffer sequence does not comprise a nucleotidesequence from the native 5′UTR sequence that is positioned 3′ tonucleotide 160 of the native CPMV 5′UTR, for example nucleotides 161 to512 as shown in FIG. 1 of Sainsbury F., and Lomonossoff G. P. (2008,Plant Physiol. 148: pp. 1212-1218; which is incorporated herein byreference), or nucleotides 161-509 of prior art CPMV HT sequence. Thatis, the incomplete M protein present in the prior art CPMV HT sequence(FIG. 1; of Sainsbury F., and Lomonossoff G. P., 2008) is removed fromthe 5′UTR in the present invention.

Plant Kozak consensus sequences are known in the art (see for exampleRangan et al. Mol. Biotechnol., 2008, July 39(3), pp. 207-213). Bothnaturally occurring and synthetic Kozak sequences may be used in theexpression enhancer or may be fused to the nucleotide sequence ofinterest as described herein.

The plant kozak sequence may be any known plant kozak sequences (see forexample L. Rangan et. al. Mol. Biotechnol. 2008), including, but notlimited to the following plant consensus sequences:

(SEQ ID NO: 2; plant kingdom) caA(A/C)a (SEQ ID NO: 3; dicots) aaA(A/C)a(SEQ ID NO: 4; arabidopsis) aa(A/G) (A/C)aThe plant kozak sequence may also be selected from the group of:

(SEQ ID NO: 5) AGAAA (SEQ ID NO: 6) AGACA (SEQ ID NO: 7) AGGAA(SEQ ID NO: 8) AAAAA (SEQ ID NO: 9) AAACA (SEQ ID NO: 10) AAGCA(SEQ ID NO: 11) AAGAA (SEQ ID NO: 12) AAAGAA (SEQ ID NO: 13) AAAGAA(SEQ ID NO: 14; Consensus sequence) (A/-)A(A/G) (A/G) (A/C)A. 

The expression enhancer CPMVX, or CPMVX+, may be operatively linked atthe 5′ end of the enhancer sequence with a regulatory region that isactive in a plant, and operatively linked to a nucleotide sequence ofinterest at the 3′ end of the expression enhancer (FIG. 6c ), in orderto drive expression of the nucleotide sequence of interest within aplant host.

CPMV HT+, CPMV HT+[WT115], CPMV HT+[511]

In another embodiment the enhancer elements is “CPMV HT+” which isdescribed in U.S. 61/971,274 (which is incorporated herein byreference). Expression enhancer “CPMV HT+” (see FIG. 6b ) comprises acomovirus 5′ untranslated region (UTR) and a modified, lengthened, ortruncated stuffer sequence.

A plant expression system comprising a first nucleic acid sequencecomprising a regulatory region, operatively linked with one or more thanone expression enhancer as described herein (e.g. CPMV HT+, CPMVHT+[WT115], CPMV HT+[511]), and a nucleotide sequence encoding arotavirus structural or nonstructural proteins is also provided.Furthermore, a nucleic acid comprising a promoter (regulatory region)sequence, an expression enhancer (e.g. CPMV HT+ or CPMV HT+[WT115])comprising a comovirus 5′UTR and a stuffer sequence with a plant kozaksequence fused to one or more nucleic acid sequences encoding arotavirus structural or nonstructural proteins are described. Thenucleic acid may further comprise a sequence comprising a comovirus 3′untranslated region (UTR), for example, a plastocyanin 3′ UTR, or other3′UTR active in a plant, and a terminator sequence, for example a NOSterminator, operatively linked to the 3′ end of the nucleotide sequenceencoding a rotavirus structural or nonstructural proteins (referred toas nucleotide of interest in FIG. 6a ), so that the nucleotide sequenceencoding the rotavirus structural or nonstructural proteins is insertedupstream from the comovirus 3′ untranslated region (UTR), plastocyanin3′ UTR, or other 3′UTR sequence.

SEQ ID NO:15 comprises a “CPMV HT” expression enhancer as known in theprior art (e.g. FIG. 1 of Sainsbury and Lomonossoff 2008, Plant Physiol.148: pp. 1212-1218; which is incorporated herein by reference). CPMV HTincludes the 5′UTR sequence from nucleotides 1-160 of SEQ ID NO:15 withmodified nucleotides at position 115 (cgt), and an incomplete M proteinwith a modified nucleotide at position 162 (acg), and lacks a plantkozak sequence (5′UTR: nucleotides 1-160; incomplete M proteinunderlined, nucleotides 161-509). SEQ ID NO:15 also includes a multiplecloning site (italics, nucleotides 510-528) which is not present in theprior art CPMV HT sequence:

SEQ ID NO: 15   1tattaaaatc ttaataggtt ttgataaaag cgaacgtggg gaaacccgaa ccaaaccttc  61ttctaaactc tctctcatct ctcttaaagc aaacttctct cttgtctttc ttgcgtgagc 121gatcttcaac gttgtcagat cgtgcttcgg caccagtaca 

ttttctt tcactgaagc 181gaaatcaaag atctctttgt ggacacgtag tgcggcgcca ttaaataacg tgtacttgtc 241ctattcttgt cggtgtggtc ttgggaaaag aaagcttgct ggaggctgct gttcagcccc 301atacattact tgttacgatt ctgctgactt tcggcgggtg caatatctct acttctgctt 361gacgaggtat tgttgcctgt acttctttct tcttcttctt gctgattggt tctataagaa 421atctagtatt ttctttgaaa cagagttttc ccgtggtttt cgaacttgga gaaagattgt 481taagcttctg tatattctqc ccaaatttg t cgggccc

CPMV HT+ with a plant kozak consensus sequence is provided in SEQ IDNO:16 (nucleotide 1-160, 5′UTR, including modified ATG at positions 115(GTG) lower case bold and italics; stuffer fragment comprising: anincomplete M protein underlined, nucleotides 161-509, with modifiednucleotide at 162 (ACG); a multiple cloning site, italics, nucleotides510-528; and a consensus plant kozak sequence, caps and bold,nucleotides 529-534).

(SEQ ID NO: 16)   1tattaaaatc ttaataggtt ttgataaaag cgaacgtggg gaaacccgaa ccaaaccttc  61ttctaaactc tctctcatct ctcttaaagc aaacttctct cttgtctttc ttgcgtgagc 121gatcttcaac gttgtcagat cgtgcttcgg caccagtaca 

ttttctt tcactgaagc 181gaaatcaaag atctctttgt ggacacgtag tgcggcgcca ttaaataacg tgtacttgtc 241ctattcttgt cggtgtggtc ttgggaaaag aaagcttgct ggaggctgct gttcagcccc 301atacattact tgttacgatt ctgctgactt tcggcgggtg caatatctct acttctgctt 361gacgaggtat tgttgcctgt acttctttct tcttcttctt gctgattggt tctataagaa 421atctagtatt ttctttgaaa cagagttttc ccgtggtttt cgaacttgga gaaagattgt 481taagcttctg tatattctgc ccaaatttg t tcgggcccaa taccgcgg (A/-)A(A/G)(A/G)(A/C)A

SEQ ID NO:17 (“CPMV HT+511”) comprises a segment of the native sequenceof the CPMV RNA 2 genome from nucleotides 1-154. The 5′UTR sequence fromnucleotides 1-511 of SEQ ID NO:17 comprises modified “atg” sequences atpositions 115 (“g” in place of “a”; italics bold) and 162 (“c” in placeof “t”; italics bold), and an incomplete M protein (underlined) fromnucleotides 161-511. CPMV HT+511 comprises a native M protein kozakconsensus sequence (nucleotides 508-511; bold):

SEQ ID NO: 17   1tattaaaatc ttaataggtt ttgataaaag cgaacgtggg gaaacccgaa ccaaaccttc  61ttctaaactc tctctcatct ctcttaaagc aaacttctct cttgtctttc ttgc

agc 121 gatcttcaac gttgtcagat cgtgcttcgg caccagtaca 

ttttctt tcactgaagc 181gaaatcaaag atctctttgt ggacacgtag tgcggcgcca ttaaataacg tgtacttgtc 241ctattcttgt cggtgtggtc ttgggaaaag aaagcttgct ggaggctgct gttcagcccc 301atacattact tgttacgatt ctgctgactt tcggcgggtg caatatctct acttctgctt 361gacgaggtat tgttgcctgt acttctttct tcttcttctt gctgattggt tctataagaa 421atctagtatt ttctttgaaa cagagttttc ccgtggtttt cgaacttgga gaaagattgt 481taagcttctg tatattctgc ccaaatttga   a ... 

Another non-limiting example of a CPMV HT+ enhancer sequence is providedby the sequence of SEQ ID NO:18 (CPMV HT+[WT115]). Expression cassettesor vectors comprising CPMV HT+ and including a plant regulatory regionin operative association with the expression enhancer sequence of SEQ IDNO: 18, and the transcriptional start site (ATG) at the 3′ end fused toa nucleotide sequence encoding rotavirus structural or nonstructuralprotein are also part o the present invention.

SEQ ID NO: 18 (CPMV HT+[WT115]) nucleotide 1-160, 5′UTR, with an ATG atposition 115-117, lower case bold; stuffer fragment comprising: anincomplete M protein underlined, nucleotides 161-509; with a modifiedATG at position 161-153 lower case bold, and underlined, a multiplecloning site, italics, nucleotides 510-528; and a plant kozak sequence,caps and bold, nucleotides 529-534).

(SEQ ID NO: 18)   1tattaaaatc ttaataggtt ttgataaaag cgaacgtggg gaaacccgaa ccaaaccttc  61ttctaaactc tctctcatct ctcttaaagc aaacttctct cttgtctttc ttgc

agc 121 gatcttcaac gttgtcagat cgtgcttcgg caccagtaca 

ttttctt tcactgaagc 181gaaatcaaag atctctttgt ggacacgtag tgcggcgcca ttaaataacg tgtacttgtc 241ctattcttgt cggtgtggtc ttgggaaaag aaagcttgct ggaggctgct gttcagcccc 301atacattact tgttacgatt ctgctgactt tcggcgggtg caatatctct acttctgctt 361gacgaggtat tgttgcctgt acttctttct tcttcttctt gctgattggt tctataagaa 421atctagtatt ttctttgaaa cagagttttc ccgtggtttt cgaacttgga gaaagattgt 481taagcttctg tatattctgc ccaaatttg t tcgggcccaa taccgcgg AG AAAA

The plant kozak sequence of SEQ ID NO:18 may be any plant kozaksequence, including but not limited, to one of the sequences of SEQ IDNO's: 2-14.

A plant expression system comprising a first nucleic acid sequencecomprising a regulatory region, operatively linked with one or more thanone expression enhancer as described herein (e.g. CPMV HT+, CPMVHT+[WT115], CPMV HT+[511]), and a nucleotide sequence encoding arotavirus structural or nonstructural protein is also provided.Furthermore, a nucleic acid comprising a promoter (regulatory region)sequence, an expression enhancer (e.g. CPMV HT+ or CPMV HT+[WT115])comprising a comovirus 5′UTR and a stuffer sequence with a plant kozaksequence fused to one or more nucleic acid sequences encoding rotavirusstructural or nonstructural protein are described. The nucleic acid mayfurther comprise a sequence comprising a comovirus 3′ untranslatedregion (UTR), for example, a plastocyanin 3′ UTR, or other 3′UTR activein a plant, and a terminator sequence, for example a NOS terminator,operatively linked to the 3′ end of the nucleotide sequence encodingrotavirus structural or nonstructural protein (referred to as nucleotideof interest in FIG. 6a ), so that the nucleotide sequence encodingrotavirus structural or nonstructural protein is inserted upstream fromthe comovirus 3′ untranslated region (UTR), plastocyanin 3′ UTR, orother 3′UTR sequence.

The occurrence of RLPs produced using the methods described herein maybe detected using any suitable method for example density gradientcentrifugation or size exclusion chromatography. RLPs may be assessedfor structure and size, for example by electron microscopy, sizeexclusion chromatography, or other techniques that would be evident toone of skill in the art.

For size exclusion chromatography, total soluble proteins may beextracted from plant tissue by homogenizing (Polytron) sample offrozen-crushed plant material in extraction buffer, and insolublematerial removed by centrifugation. Precipitation with ice cold acetoneor PEG may also be of benefit. The soluble protein is quantified, andthe extract passed through a Sephacryl™ column, for example a Sephacryl™S500 column. Blue Dextran 2000 may be used as a calibration standard.Following chromatography, fractions may be further analyzed byimmunoblot to determine the protein complement of the fraction.

The separated fraction may be for example a supernatant (if centrifuged,sedimented, or precipitated), or a filtrate (if filtered), and isenriched for proteins, or suprastructure proteins, and include highermolecular weight, particles such as single-layered (sl), double-layered(dl) or triple-layered (tl) RLPs.

The separated fraction may be further processed to isolate, purify,concentrate or a combination thereof, the proteins, suprastructureproteins or higher-order particles by, for example, additionalcentrifugation steps, precipitation, chromatographic steps (e.g. sizeexclusion, ion exchange, affinity chromatography), tangential flowfiltration, or a combination thereof. The presence of purified proteins,suprastructure proteins or higher-order particles such as RLPs, may beconfirmed by, for example, native or SDS-PAGE, Western analysis using anappropriate detection antibody, capillary electrophoresis, electronmicroscopy, or any other method as would be evident to one of skill inthe art.

The RLP's produced according to the present invention may be purified,partially purified from a plant, portion of a plant or plant matter, ormay be administered as an oral vaccine, using methods as know to one ofskill in the art.

RLP purification may involve gradient centrifugation, for examplesucrose, iodixanol, OptiPrep™ or cesium chloride (CsCl) densitygradients may be used to purify or partially purify the RLPs fromtransformed plant biomass. As shown for example in FIG. 4, an iodixanolstep gradient or iodixanol continuous gradient might be used to purifythe RLP and/or expressed rotavirus structural proteins.

Calcium (Ca2+) concentration has been shown to be important for thetriple-layer particle (TLP) to double layer particle (DLP)transformation and is strain dependent (see for example Martin et al.Journal of Virology, January 2002, which is incorporated herein byreference). Complete loss of the outer-capsid proteins from TLPs (TLPdecapsidation) takes place in the nanomolar range of [Ca2+]. Thereforethe extraction and/or purification of RLP may be performed in thepresence of calcium, and the step of gradient centrifugation may beperformed in the presence of calcium, for example in the present ofCaCl2. The concentration of CaCl2 maybe between for example, 1 mM and1000 mM, or any amount there between, such as 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300,350, 400, 450, 500, 50, 600, 650, 700, 750, 800, 850, 900, 950 mM or anyamount therebetween.

The plants, or plant fragments may be minimally processed. By the term“minimal processing” it is meant plant matter, for example, a plant orportion thereof comprising a protein of interest and/or the RLP which ispartially purified to yield a plant extract, homogenate, fraction ofplant homogenate or the like (i.e. minimally processed). Partialpurification may comprise, but is not limited to disrupting plantcellular structures thereby creating a composition comprising solubleplant components, and insoluble plant components which may be separatedfor example, but not limited to, by centrifugation, filtration or acombination thereof. In this regard, proteins secreted within theextracellular space of leaf or other tissues could be readily obtainedusing vacuum or centrifugal extraction, or tissues could be extractedunder pressure by passage through rollers or grinding or the like tosqueeze or liberate the protein free from within the extracellularspace. Minimal processing could also involve preparation of crudeextracts of soluble proteins, since these preparations would havenegligible contamination from secondary plant products. Further, minimalprocessing may involve aqueous extraction of soluble protein fromleaves, followed by precipitation with any suitable salt. Other methodsmay include large scale maceration and juice extraction in order topermit the direct use of the extract. The RLPs may be purified orextracted using any suitable method for example mechanical orbiochemical extraction.

The one or more rotavirus structural protein may be synthesized at anamount up to 2 g per kilogram of plant fresh weight. For example, theamount of synthesized structural protein maybe between 1 and 2 g perkilogram of fresh weight, or any amount there between, such as 1.0, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 g per kilogram of fresh weightor any amount therebetween. For example, the structural protein may besynthesized at an amount up to 1.54 g per kilogram of plant freshweight.

The size (i.e. the diameter) of the above-defined RLPs, maybe measuresfor example by dynamic light scattering (DLS) or electron microscope(EM) techniques, is usually between 50 to 110 nm, or any sizetherebetween. For example, the size of the intact RLP structure mayrange from about 70 nm to about 110 nm, or any size therebetween, suchas 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm or any sizetherebetween.

Nucleotide Sequences

The present invention further provides a nucleic acid comprising anucleotide sequence encoding one or more rotavirus structural proteinoperatively linked to a regulatory region active in a plant. Thenucleotide sequence may be optimized for example for human codon usageor plant codon usage. Furthermore one or more rotavirus structuralprotein may be operatively linked to one or more than one amplificationelements. In addition one or more rotavirus structural protein may beoperatively linked to one or more than one compartment targetingsequence. The one or more rotavirus structural protein encoded by thenucleotide sequence may be for example VP2, VP4, VP6 or VP7. Furthermorethe one or more rotavirus structural protein encoded by the nucleotidesequence may be for example from any rotavirus group A to G, but morepreferably from rotavirus group A. Furthermore, the one or morerotavirus structural protein encoded by the nucleotide sequence maybefrom any rotavirus strain having a genotype of any combinations of G-and P-types from G1 to G27 and from P1 to P34, and more preferably fromG1 to G19 and from P1 to P27, including, but not limited to G1P[8],G2P[4], G2P[8], G3P[8], G4P[8], G9P[6], G9P[8], rotavirus A WA strain,rotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] strain orrotavirus SA11 strain.

A nucleic acid sequence referred to in the present invention, may be“substantially homologous”, “substantially similar” or “substantiallyidentical” to a sequence, or a compliment of the sequence if the nucleicacid sequence hybridize to one or more than one nucleotide sequence or acompliment of the nucleic acid sequence as defined herein understringent hybridization conditions. Sequences are “substantiallyhomologous” “substantially similar” “substantially identical” when atleast about 70%, or between 70 to 100%, or any amount therebetween, forexample 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100%, or any amount therebetween, of the nucleotides match over adefined length of the nucleotide sequence providing that such homologoussequences exhibit one or more than one of the properties of thesequence, or the encoded product as described herein.

For example the present invention provides an isolated polynucleotidecomprising a nucleotide sequence which encodes one or more rotavirusprotein, for example a structural or nonstructural rotavirus protein,that is at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% 100% or any amounttherebetween identical to sequences as defines for example in SEQ IDNOs: 21, 27, 32, 37 or 42. The polynucleotide may be human codonoptimized by any of the methods known in the art. The nucleotidesequence may enclode for example a rotavirus protein that is at least60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% 100% or any amount therebetween identicalthe amino acid sequence of SEQ ID NOs: 24, 29, 34, 39 or 44.

Furthermore, the present invention provides RLPS that comprise rotavirusstructural proteins that are for example encoded by nucleic acids thatare at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% 100% or any amount therebetweenidentical to sequences as defines for example in SEQ ID NOs: 21, 27, 32,37 or 42.

Such a sequence similarity or identity may be determined using anucleotide sequence comparison program, such as that provided withinDNASIS (using, for example but not limited to, the following parameters:GAP penalty 5, # of top diagonals 5, fixed GAP penalty 10, k tuple 2,floating gap 10, and window size 5). However, other methods of alignmentof sequences for comparison are well-known in the art for example thealgorithms of Smith & Waterman (1981, Adv. Appl. Math. 2:482), Needleman& Wunsch (J. Mol. Biol. 48:443, 1970), Pearson & Lipman (1988, Proc.Nat'l. Acad. Sci. USA 85:2444), and by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and BLAST, available through theNIH.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology, Ausubel et al., eds. 1995 supplement),or using Southern or Northern hybridization under stringent conditions(see Maniatis et al., in Molecular Cloning (A Laboratory Manual), ColdSpring Harbor Laboratory, 1982). Preferably, sequences that aresubstantially homologous exhibit at least about 80% and most preferablyat least about 90% sequence similarity over a defined length of themolecule.

An example of one such stringent hybridization conditions may beovernight (from about 16-20 hours) hybridization in 4×SSC at 65° C.,followed by washing in 0.1×SSC at 65° C. for an hour, or 2 washes in0.1×SSC at 65° C. each for 20 or 30 minutes. Alternatively an exemplarystringent hybridization condition could be overnight (16-20 hours) in50% formamide, 4×SSC at 42° C., followed by washing in 0.1×SSC at 65° C.for an hour, or 2 washes in 0.1×SSC at 65° C. each for 20 or 30 minutes,or overnight (16-20 hours), or hybridization in Church aqueous phosphatebuffer (7% SDS; 0.5M NaPO4 buffer pH 7.2; 10 mM EDTA) at 65° C., with 2washes either at 50° C. in 0.1×SSC, 0.1% SDS for 20 or 30 minutes each,or 2 washes at 65° C. in 2×SSC, 0.1% SDS for 20 or 30 minutes each forunique sequence regions.

A nucleic acid encoding a rotavirus structural polypeptide may bedescribed as a “rotavirus nucleic acid”, a “rotavirus nucleotidesequence”, a “rotavirus nucleic acid”, or a “rotavirus nucleotidesequence”. For example, which is not to be considered limiting, avirus-like particle comprising one or more rotavirus structural proteinor rotavirus structural polypeptide, may be described as a “rotavirusVLP”, “RVLP” or “RLP”.

Many organisms display a bias for use of particular codons to code forinsertion of a particular amino acid in a growing peptide chain. Codonpreference or codon bias, differences in codon usage between organisms,is afforded by degeneracy of the genetic code, and is well documentedamong many organisms. Codon bias often correlates with the efficiency oftranslation of messenger RNA (mRNA), which is in turn believed to bedependent on, inter alia, the properties of the codons being translatedand the availability of particular transfer RNA (tRNA) molecules. Thepredominance of selected tRNAs in a cell is generally a reflection ofthe codons used most frequently in peptide synthesis. Accordingly, genescan be tailored for optimal gene expression in a given organism based oncodon optimization. The process of optimizing the nucleotide sequencecoding for a heterologously expressed protein can be an important stepfor improving expression yields. The optimization requirements mayinclude steps to improve the ability of the host to produce the foreignprotein.

“Codon optimization” is defined as modifying a nucleic acid sequence forenhanced expression in cells of interest by replacing at least one, morethan one, or a significant number, of codons of the native sequence withcodons that may be more frequently or most frequently used in the genesof another organism or species. Various species exhibit particular biasfor certain codons of a particular amino acid.

The present invention includes synthetic polynucleotide sequences thathave been codon optimized for example the sequences have been optimizedfor human codon usage or plant codon usage. The codon optimizedpolynucleotide sequences may then be expressed in plants. Morespecifically the sequences optimized for human codon usage or plantcodon usage may be expressed in plants. Without wishing to be bound bytheory, it is believed that the sequences optimized for human codonincreases the guanine-cytosine content (GC content) of the sequence andimproves expression yields in plants.

There are different codon-optimisation techniques known in the art forimproving, the translational kinetics of translationally inefficientprotein coding regions. These techniques mainly rely on identifying thecodon usage for a certain host organism. If a certain gene or sequenceshould be expressed in this organism, the coding sequence of such genesand sequences will then be modified such that one will replace codons ofthe sequence of interest by more frequently used codons of the hostorganism.

Amino Acid Sequences

Non-limiting examples of rotavirus structural protein are rotavirusprotein VP2, VP4, VP6 and VP7, and a fragment of VP2, VP4, VP6 and VP7.Non-limiting examples of VP2, VP4, VP6 and VP7, or fragments of VP2,VP4, VP6 and VP7 protein that may be used according to the presentinvention include those VP2, VP4 VP6 and VP7 protein from rotavirusstrain G9 P[6], rotavirus A WA strain, rotavirus A vaccineUSA/Rotarix-A41CB052A/1988/G1P1A[8] strain and rotavirus SA11 strain.For example, but not limited to Rotarix-A41CB052A: VP4 (accession#JN849113), VP7: (accession #JN849114), rotavirus A WA strain: VP2(accession #X14942), VP4: (accession #L34161), VP6 (accession #K02086),VP7: (accession #GU723327), NSP4 (accession #K02032), rotavirus SA11strain: VP2 (accession #NC_011506), VP4 (accession #NC_011510), VP6(accession #NC_011509), VP7 (accession #NC_011503) and NSP4 (accession#NC_011504).

An example of a VP2 structural protein, which is not to be consideredlimiting, is set forth in the amino acid sequence of SEQ ID NO: 24.Furthermore, the VP2 structural protein may comprise the sequence setforth in SEQ ID NO: 24, or a sequence having at least about 90-100%sequence similarity thereto, including any percent similarity withinthese ranges, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% sequencesimilarity thereto. In addition, a VP2 structural protein may be encodedby a nucleotide sequence as set forth in SEQ ID NO:21 or a sequencehaving at least about 80-100% sequence similarity thereto, including anypercent similarity within these ranges, such as 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similaritythereto.

An example of a VP4 structural protein, which is not to be consideredlimiting, is set forth in the amino acid sequence of SEQ ID NO: 34.Furthermore, the VP4 structural protein may comprise the sequence setforth in SEQ ID NO: 34, or a sequence having at least about 90-100%sequence similarity thereto, including any percent similarity withinthese ranges, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% sequencesimilarity thereto. In addition, a VP4 structural protein may be encodedby a nucleotide sequence as set forth in SEQ ID NO: 32 or a sequencehaving at least about 80-100% sequence similarity thereto, including anypercent similarity within these ranges, such as 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similaritythereto.

An example of a VP6 structural protein, which is not to be consideredlimiting, is set forth in the amino acid sequence of SEQ ID NO: 29.Furthermore, the VP6 structural protein may comprise the sequence setforth in SEQ ID NO: 29, or a sequence having at least about 90-100%sequence similarity thereto, including any percent similarity withinthese ranges, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% sequencesimilarity thereto. In addition, a VP6 structural protein may be encodedby a nucleotide sequence as set forth in SEQ ID NO:27 or a sequencehaving at least about 80-100% sequence similarity thereto, including anypercent similarity within these ranges, such as 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similaritythereto.

An example of a VP7 structural protein, which is not to be consideredlimiting, is set forth in the amino acid sequence of SEQ ID NO: 39.Furthermore, the VP7 structural protein may comprise the sequence setforth in SEQ ID NO: 39, or a sequence having at least about 90-100%similarity thereto, including any percent similarity within theseranges, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similaritythereto. In addition, a VP7 structural protein may be encoded by anucleotide sequence as set forth in SEQ ID NO:37 or a sequence having atleast about 80-100% sequence similarity thereto, including any percentsimilarity within these ranges, such as 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similarity thereto.

An example of a NSP4 structural protein, which is not to be consideredlimiting, is set forth in the amino acid sequence of SEQ ID NO: 44.Furthermore, the NSP4 nonstructural protein may comprise the sequenceset forth in SEQ ID NO: 44, or a sequence having at least about 90-100%similarity thereto, including any percent similarity within theseranges, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similaritythereto. In addition, a NSP4 nonstructural protein may be encoded by anucleotide sequence as set forth in SEQ ID NO: 42 or a sequence havingat least about 80-100% sequence similarity thereto, including anypercent similarity within these ranges, such as 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similaritythereto.

Amino acid sequence similarity or identity may be computed by using theBLASTP and TBLASTN programs which employ the BLAST (basic localalignment search tool) 2.0 algorithm. Techniques for computing aminoacid sequence similarity or identity are well known to those skilled inthe art, and the use of the BLAST algorithm is described in ALTSCHUL etal. (1990, J Mol. Biol. 215: 403-410) and ALTSCHUL et al. (1997, NucleicAcids Res. 25: 3389-3402).

Without wishing to be bound by theory, the protein concentration andratio of the different rotavirus structural proteins may be importantfor the assembly efficiency of RLPs. Therefore multiplicity and time ofinfection, may be important to manipulate protein concentration and theoverall assembly efficiency of RLPs in plants.

The construct of the present invention may be transiently expressed in aplants or portion of a plant. A transient expression system relying onthe epichromosomal expression of recombinant Agrobacterium tumefaciensin a plant, portion of a plant or plant cell may be used to express therotavirus structural protein, targeted to various cell compartments orsub-compartments. A transient expression system allows for a highproduction speed. Furthermore, large amounts of protein can be attainedwithin a few days after infiltration of recombinant Agrobacterium inplants (Rybicki, 2010; Fischer et al., 1999). It is also possible toexpress long gene sequences and have more than one gene simultaneouslyexpressed in the same cell, allowing for efficient assembly ofmultimeric proteins (Lombardi et al., 2009).

The nucleotide sequences encoding for the rotavirus structural proteinsand nonstructural proteins may be transferred into the plant host using1, 2, 3, 4 or 5 transformed Agrobacterium tumefaciens strains (asdescribed in Table 1 and accompanying text

During transient expression post-transcriptional gene silencing maylimit the expression of the heterologous proteins in plants. Theco-expression of a suppressor of silencing, for example, but not limitedto Nss from Tomato spotted wilt virus may be used to counteract thespecific degradation of transgene mRNAs (Brigneti et al., 1998).Alternate suppressors of silencing are well known in the art and may beused as described herein (Chiba et al., 2006, Virology 346:7-14; whichis incorporated herein by reference), for example but not limited toHcPro, TEV-p1/HC-Pro (Tobacco etch virus-p1/HC-Pro), BYV-p21, p19 ofTomato bushy stunt virus (TBSV p19), capsid protein of Tomato crinklevirus (TCV-CP), 2b of Cucumber mosaic virus; CMV-2b), p25 of Potatovirus X (PVX-p25), p11 of Potato virus M (PVM-p11), p11 of Potato virusS (PVS-p11), p16 of Blueberry scorch virus, (BScV-p16), p23 of Citrustristexa virus (CTV-p23), p24 of Grapevine leafroll-associated virus-2,(GLRaV-2 p24), p10 of Grapevine virus A, (GVA-p10), p14 of Grapevinevirus B (GVB-p14), p10 of Heracleum latent virus (HLV-p10), or p16 ofGarlic common latent virus (GCLV-p16). Therefore, a suppressor ofsilencing, for example HcPro, TEV-p1/HC-Pro, BYV-p21, TBSV p19, TCV-CP,CMV-2b, PVX-p25, PVM-p11, PVS-p11, BScV-p16, CTV-p23, GLRaV-2 p24,GBV-p14, HLV-p10, GCLV-p16or GVA-p10, may be co-expressed along with oneor more rotavirus structural or non structural protein for example VP2,VP4, VP6, VP7 and NSP4 or a combination thereof, to further ensure highlevels of protein production within a plant or portion of a plant.

The present invention also provides a methods as described above,wherein an additional (second, third, fourth, fifth or sixth) nucleotidesequence is expressed within the plant, the additional (second, third,fourth, fifth or sixth) nucleotide sequence encoding a suppressor ofsilencing is operatively linked with an additional (second, third,fourth, fifth or sixth) regulatory region that is active in the plant.The nucleotide sequence encoding a suppressor of silencing may be, forexample Nss, HcPro, TEV-p1/HC-Pro, BYV-p21, TBSV p19, TCV-CP, CMV-2b,PVX-p25, PVM-p11, PVS-p11, BScV-p16, CTV-p23, GLRaV-2 p24, GBV-p14,HLV-p10, GCLV-p16 or GVA-p10.

As described below, transient expression methods may be used to expressthe constructs of the present invention (see Liu and Lomonossoff, 2002,Journal of Virological Methods, 105:343-348; which is incorporatedherein by reference). Alternatively, a vacuum-based transient expressionmethod, as described by Kapila et al., 1997, which is incorporatedherein by reference) may be used. These methods may include, forexample, but are not limited to, a method of Agro-inoculation orAgro-infiltration, syringe infiltration, however, other transientmethods may also be used as noted above. With Agro-inoculation,Agro-infiltration, or syringe infiltration, a mixture of Agrobacteriacomprising the desired nucleic acid enter the intercellular spaces of atissue, for example the leaves, aerial portion of the plant (includingstem, leaves and flower), other portion of the plant (stem, root,flower), or the whole plant. After crossing the epidermis theAgrobacteria infect and transfer t-DNA copies into the cells. The t-DNAis episomally transcribed and the mRNA translated, leading to theproduction of the protein of interest in infected cells, however, thepassage of t-DNA inside the nucleus is transient.

To aid in identification of transformed plant cells, the constructs ofthis invention may be further manipulated to include plant selectablemarkers. Useful selectable markers include enzymes that provide forresistance to chemicals such as an antibiotic for example, gentamycin,hygromycin, kanamycin, or herbicides such as phosphinothrycin,glyphosate, chlorosulfuron, and the like. Similarly, enzymes providingfor production of a compound identifiable by colour change such as GUS(beta-glucuronidase), or luminescence, such as luciferase or GFP, may beused.

Also considered part of this invention are transgenic plants, plantcells or seeds containing the constructs as described herein. Methods ofregenerating whole plants from plant cells are also known in the art. Ingeneral, transformed plant cells are cultured in an appropriate medium,which may contain selective agents such as antibiotics, where selectablemarkers are used to facilitate identification of transformed plantcells. Once callus forms, shoot formation can be encouraged by employingthe appropriate plant hormones in accordance with known methods and theshoots transferred to rooting medium for regeneration of plants. Theplants may then be used to establish repetitive generations, either fromseeds or using vegetative propagation techniques. Transgenic plants canalso be generated without using tissue cultures.

The use of the terms “regulatory region”, “regulatory element” or“promoter” in the present application is meant to reflect a portion ofnucleic acid typically, but not always, upstream of the protein codingregion of a gene, which may be comprised of either DNA or RNA, or bothDNA and RNA. When a regulatory region is active, and in operativeassociation, or operatively linked, with a gene of interest, this mayresult in expression of the gene of interest. A regulatory element maybe capable of mediating organ specificity, or controlling developmentalor temporal gene activation. A “regulatory region” may includes promoterelements, core promoter elements exhibiting a basal promoter activity,elements that are inducible in response to an external stimulus,elements that mediate promoter activity such as negative regulatoryelements or transcriptional enhancers. “Regulatory region”, as usedherein, may also includes elements that are active followingtranscription, for example, regulatory elements that modulate geneexpression such as translational and transcriptional enhancers,translational and transcriptional repressors, upstream activatingsequences, and mRNA instability determinants. Several of these latterelements may be located proximal to the coding region.

In the context of this disclosure, the term “regulatory element” or“regulatory region” typically refers to a sequence of DNA, usually, butnot always, upstream (5′) to the coding sequence of a structural gene,which controls the expression of the coding region by providing therecognition for RNA polymerase and/or other factors required fortranscription to start at a particular site. However, it is to beunderstood that other nucleotide sequences, located within introns, or3′ of the sequence may also contribute to the regulation of expressionof a coding region of interest. An example of a regulatory element thatprovides for the recognition for RNA polymerase or other transcriptionalfactors to ensure initiation at a particular site is a promoter element.Most, but not all, eukaryotic promoter elements contain a TATA box, aconserved nucleic acid sequence comprised of adenosine and thymidinenucleotide base pairs usually situated approximately 25 base pairsupstream of a transcriptional start site. A promoter element comprises abasal promoter element, responsible for the initiation of transcription,as well as other regulatory elements (as listed above) that modify geneexpression.

There are several types of regulatory regions, including those that aredevelopmentally regulated, inducible or constitutive. A regulatoryregion that is developmentally regulated, or controls the differentialexpression of a gene under its control, is activated within certainorgans or tissues of an organ at specific times during the developmentof that organ or tissue. However, some regulatory regions that aredevelopmentally regulated may preferentially be active within certainorgans or tissues at specific developmental stages, they may also beactive in a developmentally regulated manner, or at a basal level inother organs or tissues within the plant as well. Examples oftissue-specific regulatory regions, for example see-specific aregulatory region, include the napin promoter, and the cruciferinpromoter (Rask et al., 1998, J. Plant Physiol. 152: 595-599; Bilodeau etal., 1994, Plant Cell 14: 125-130). An example of a leaf-specificpromoter includes the plastocyanin promoter (see U.S. Pat. No.7,125,978, which is incorporated herein by reference).

An inducible regulatory region is one that is capable of directly orindirectly activating transcription of one or more DNA sequences orgenes in response to an inducer. In the absence of an inducer the DNAsequences or genes will not be transcribed. Typically the protein factorthat binds specifically to an inducible regulatory region to activatetranscription may be present in an inactive form, which is then directlyor indirectly converted to the active form by the inducer. However, theprotein factor may also be absent. The inducer can be a chemical agentsuch as a protein, metabolite, growth regulator, herbicide or phenoliccompound or a physiological stress imposed directly by heat, cold, salt,or toxic elements or indirectly through the action of a pathogen ordisease agent such as a virus. A plant cell containing an inducibleregulatory region may be exposed to an inducer by externally applyingthe inducer to the cell or plant such as by spraying, watering, heatingor similar methods. Inducible regulatory elements may be derived fromeither plant or non-plant genes (e.g. Gatz, C. and Lenk, L R. P., 1998,Trends Plant Sci. 3, 352-358; which is incorporated by reference).Examples, of potential inducible promoters include, but not limited to,tetracycline-inducible promoter (Gatz, C., 1997, Ann. Rev. PlantPhysiol. Plant Mol. Biol. 48, 89-108; which is incorporated byreference), steroid inducible promoter (Aoyama. T. and Chua, N. H.,1997, Plant 1. 2, 397-404; which is incorporated by reference) andethanol-inducible promoter (Salter, M. G., et al, 1998, Plant Journal16, 127-132; Caddick, M. X., et al, 1998, Nature Biotech. 16, 177-180,which are incorporated by reference) cytokinin inducible IB6 and CKI 1genes (Brandstatter, I. and Kieber, 1.1., 1998, Plant Cell 10,1009-1019; Kakimoto, T., 1996, Science 274, 982-985; which areincorporated by reference) and the auxin inducible element, DR5(Ulmasov, T., et al., 1997, Plant Cell 9, 1963-1971; which isincorporated by reference).

A constitutive regulatory region directs the expression of a genethroughout the various parts of a plant and continuously throughoutplant development. Examples of known constitutive regulatory elementsinclude promoters associated with the CaMV 35S transcript (Odell et al.,1985, Nature, 313: 810-812), the rice actin 1 (Zhang et al, 1991, PlantCell, 3: 1155-1165), actin 2 (An et al., 1996, Plant J., 10: 107-121),or tms 2 (U.S. Pat. No. 5,428,147, which is incorporated herein byreference), and triosephosphate isomerase 1 (Xu et. al., 1994, PlantPhysiol. 106: 459-467) genes, the maize ubiquitin 1 gene (Cornejo et al,1993, Plant Mol. Biol. 29: 637-646), the Arabidopsis ubiquitin 1 and 6genes (Holtorf et al, 1995, Plant Mol. Biol. 29: 637-646), and thetobacco translational initiation factor 4A gene (Mandel et al, 1995,Plant Mol. Biol. 29: 995-1004).

The term “constitutive” as used herein does not necessarily indicatethat a gene under control of the constitutive regulatory region isexpressed at the same level in all cell types, but that the gene isexpressed in a wide range of cell types even though variation inabundance is often observed. Constitutive regulatory elements may becoupled with other sequences to further enhance the transcription and/ortranslation of the nucleotide sequence to which they are operativelylinked. For example, the CPMV-HT system is derived from the untranslatedregions of the Cowpea mosaic virus (CPMV) and demonstrates enhancedtranslation of the associated coding sequence. By “native” it is meantthat the nucleic acid or amino acid sequence is naturally occurring, or“wild type”. By “operatively linked” it is meant that the particularsequences, for example a regulatory element and a coding region ofinterest, interact either directly or indirectly to carry out anintended function, such as mediation or modulation of gene expression.The interaction of operatively linked sequences may, for example, bemediated by proteins that interact with the operatively linkedsequences.

The RLP produced within a plant may produce a rotavirus VP7 structuralprotein comprising plant-specific N-glycans. Therefore, this inventionalso provides for a RLP comprising VP7 having plant specific N-glycans.

Furthermore, modification of N-glycan in plants is known (see forexample U.S. 60/944,344; which is incorporated herein by reference) andVP7 having modified N-glycans may be produced. VP7 comprising a modifiedglycosylation pattern, for example with reduced fucosylated,xylosylated, or both, fucosylated and xylosylated, N-glycans may beobtained, or VP7 having a modified glycosylation pattern may beobtained, wherein the protein lacks fucosylation, xylosylation, or both,and comprises increased galactosylation. Furthermore, modulation ofpost-translational modifications, for example, the addition of terminalgalactose may result in a reduction of fucosylation and xylosylation ofthe expressed VP7 when compared to a wild-type plant expressing VP7.

For example, which is not to be considered limiting, the synthesis ofVP7 having a modified glycosylation pattern may be achieved byco-expressing VP7 along with a nucleotide sequence encoding beta-1.4galactosyltransferase (GalT), for example, but not limited to mammalianGalT, or human GalT however GalT from another sources may also be used.The catalytic domain of GalT may also be fused to a CTS domain (i.e. thecytoplasmic tail, transmembrane domain, stem region) ofN-acetylglucosaminyl transferase (GNT1), to produce a GNT1-GalT hybridenzyme, and the hybrid enzyme may be co-expressed with VP7. The VP7 mayalso be co-expressed along with a nucleotide sequence encodingN-acetylglucosaminyl transferase III (GnT-III), for example but notlimited to mammalian GnT-III or human GnT-III, GnT-III from othersources may also be used. Additionally, a GNT1-GnT-III hybrid enzyme,comprising the CTS of GNT1 fused to GnT-III may also be used.

Therefore the present invention also provides RLPs comprising VP7 havingmodified N-glycans.

Without wishing to be bound by theory, the presence of plant N-glycanson VP7 may stimulate the immune response by promoting the binding of VP7by antigen presenting cells. Stimulation of the immune response usingplant N glycan has been proposed by Saint-Jore-Dupas et al. (2007).

Table 2 lists sequences provided in various embodiments of theinvention.

TABLE 2 SEQ ID NO Description Page/FIG. 1 expression enhancer CPMVX 2plant kingdom kozak consensus sequence 3 Dicots kozak consensus sequence4 Arabidopsis kozak consensus sequence 5-13 plant kozak sequences 14Kozak consensus sequence 15 CPMV HT 16 CPMV HT+ 17 CPMV HT+ 511 18 CPMVHT+ [WT115] 19 IF-WA_VP2(opt).s1 + 3c FIG. 7A 20 IF-WA_VP2(opt).s1 − 4rFIG. 7B 21 Optimized coding sequence of Rotavirus A VP2 FIG. 7C fromstrain WA 22 Construct 1191 FIG. 7E 23 Expression cassette number 1710FIG. 7F 24 Amino acid sequence of VP2 from Rotavirus A WA FIG. 7G strain25 IF-WA_VP6(opt).s1 + 3c FIG. 8A 26 IF-WA_VP6(opt).s1 − 4r FIG. 8B 27Optimized coding sequence of Rotavirus A VP6 FIG. 8C from strain WA 28Expression cassette number 1713 FIG. 8D 29 Amino acid sequence of VP6from Rotavirus A WA FIG. 8E strain 30 IF-Rtx_VP4(opt).s1 + 3c FIG. 9A 31IF-Rtx_VP4(opt).s1 − 4r FIG. 9B 32 Optimized coding sequence ofRotavirus A VP4 FIG. 9C from strain RVA/Vaccine/USA/Rotarix-A41CB052A/1988/G1P1A[8] 33 Expression cassette number 1730 FIG. 9D 34Amino acid sequence of VP4 from Rotavirus A FIG. 9E Rotarix strain 35IF-TrSP + Rtx_VP7(opt).s1 + 3c FIG. 10A 36 IF-Rtx_VP7(opt).s1 − 4r FIG.10B 37 Optimized coding sequence of Rotavirus A VP7 FIG. 10C from strainRVA/Vaccine/USA/Rotarix- A41CB052A/1988/G1P1A[8] 38 Expression cassettenumber 1734 FIG. 10D 39 Amino acid sequence of TrSp-VP7 from RotavirusFIG. 10E A vaccine USA/Rotarix- A41CB052A/1988/G1P1A[8] strain 40IF-WA_NSP4.s1 + 3c FIG. 11A 41 IF-WA_NSP4.s1 − 4r FIG. 11B 42 Codingsequence of Rotavirus A NSV4 from strain FIG. 11C WA 43 Expressioncassette number 1706 FIG. 11D 44 Amino acid sequence of NSP4 fromRotavirus A FIG. 11E WA strain 45 IF(C160)-WA_VP2(opt).c FIG. 12A 46Construct 1190 FIG. 12C 47 Expression cassette number 1108 FIG. 12D 48IF(C160)-WA_VP6(opt).c FIG. 13A 49 Expression cassette number 1128 FIG.13B 50 IF(C160)-Rtx_VP4(opt).c FIG. 14A 51 Expression cassette number1178 FIG. 14B 52 IF(C160)-TrSP + Rtx_VP7(opt).c FIG. 15A 53 Expressioncassette number 1199 FIG. 15B

The present invention will be further illustrated in the followingexamples.

EXAMPLES Example 1 Materials and Methods

TABLE 3 Constructs De- De- Constr. # scription FIG. Constr.# scriptionFIG. 1108 160-VP2 3A/3B 1706 CPMV-HT 3A/3B NSP4 4A/4B 1128 160-VP6 3A/3B1708 CPMV-HT 3A/3B VP6/2 4A/4B 1178 160-VP4 3A/3B 2408 160-VP7/4 3A/3B4A/4B 1199 160-VP7 3A/3B 1769 CPMV-HT 3A/3B/5 VP7/4/6/2 1710 CPMV-HT4A/4B 2441 CPMV-HT 5 VP2 VP4/7/ NSP4/6/2 1713 CPMV-HT 4A/4B 2400160-VP6/2 4A/4B VP6 1730 CPMV-HT 4A/4B 1719 CPMV-HT 4A/4B VP4 VP7/4 1734CPMV-HT 4A/4B VP71. 2X35S/CPMV-HT/RVA(WA) VP2(opt)/NOS (Construct Number 1710)

An optimized sequence encoding VP2 from Rotavirus A WA strain was clonedinto 2X35S-CPMV-HT-NOS expression system in a plasmid containingPlasto_pro/P19/Plasto_ter expression cassette using the followingPCR-based method. A fragment containing the VP2 coding sequence wasamplified using primers IF-WA_VP2(opt).s1+3c (FIG. 7a , SEQ ID NO: 19)and IF-WA_VP2(opt).s1−4r (FIG. 7B, SEQ ID NO: 20), using optimized VP2gene sequence (FIG. 7C, SEQ ID NO:21) as template. For sequenceoptimization, VP2 protein sequence (Genbank accession number CAA33074)was backtranslated and optimized for human codon usage, GC content andmRNA structure. The PCR product was cloned in 2X35S/CPMV-HT/NOSexpression system using In-Fusion cloning system (Clontech, MountainView, Calif.). Construct number 1191 (FIG. 7D) was digested with SacIIand StuI restriction enzyme and the linearized plasmid was used for theIn-Fusion assembly reaction. Construct number 1191 is an acceptorplasmid intended for “In Fusion” cloning of genes of interest in aCPMV-HT-based expression cassette. It also incorporates a gene constructfor the co-expression of the TBSV P19 suppressor of silencing under thealfalfa Plastocyanin gene promoter and terminator. The backbone is apCAMBIA binary plasmid and the sequence from left to right t-DNA bordersis presented in FIG. 7E (SEQ ID NO: 22). The resulting construct wasgiven number 1710 (FIG. 7F, SEQ ID NO: 23). The amino acid sequence ofVP2 from Rotavirus A strain WA is presented in FIG. 7G (SEQ ID NO: 24).A representation of plasmid 1710 is presented in FIG. 7H.

2. 2X35S/CPMV-HT/RVA(WA) VP6(opt)/NOS (Construct Number 1713)

An optimized sequence encoding VP6 from Rotavirus A WA strain was clonedinto 2X35S-CPMV-HT-NOS expression system in a plasmid containingPlasto_pro/P19/Plasto_ter expression cassette using the followingPCR-based method. A fragment containing the VP6 coding sequence wasamplified using primers IF-WA_VP6(opt).s1+3c (FIG. 8A, SEQ ID NO: 25)and IF-WA_VP6(opt).s1−4r (FIG. 8B, SEQ ID NO: 26), using optimized VP6gene sequence (FIG. 8C, SEQ ID NO:27) as template. For sequenceoptimization, VP6 protein sequence (Genbank accession number AAA47311)was backtranslated and optimized for human codon usage, GC content andmRNA structure. The PCR product was cloned in 2X35S/CPMV-HT/NOSexpression system using In-Fusion cloning system (Clontech, MountainView, Calif.). Construct number 1191 (FIG. 7D) was digested with SacIIand StuI restriction enzyme and the linearized plasmid was used for theIn-Fusion assembly reaction. Construct number 1191 is an acceptorplasmid intended for “In Fusion” cloning of genes of interest in aCPMV-HT-based expression cassette. It also incorporates a gene constructfor the co-expression of the TBSV P19 suppressor of silencing under thealfalfa Plastocyanin gene promoter and terminator. The backbone is apCAMBIA binary plasmid and the sequence from left to right t-DNA bordersis presented in FIG. 7E (SEQ ID NO: 22). The resulting construct wasgiven number 1713 (FIG. 8D, SEQ ID NO: 28). The amino acid sequence ofVP6 from Rotavirus A strain WA is presented in FIG. 8E (SEQ ID NO: 29).A representation of plasmid 1713 is presented in FIG. 8F.

3. 2X35S/CPMV-HT/RVA(Rtx) VP4(opt)/NOS (Construct Number 1730)

An optimized sequence encoding VP4 from Rotavirus A vaccineUSA/Rotarix-A41CB052A/1988/G1P1A[8] strain was cloned into2X35S/CPMV-HT/NOS in a plasmid containing Plasto_pro/P19/Plasto_terexpression cassette using the following PCR-based method. A fragmentcontaining the VP4 coding sequence was amplified using primersIF-Rtx_VP4(opt).s1+3c (FIG. 9A, SEQ ID NO: 30) and IF-Rtx_VP4(opt).s1−4r(FIG. 9B, SEQ ID NO: 31), using optimized VP4 gene sequence (FIG. 9C,SEQ ID NO: 32) as template. For sequence optimization, VP4 proteinsequence (Genbank accession number AEX30660) was backtranslated andoptimized for human codon usage, GC content and mRNA structure. The PCRproduct was cloned in 2X35S/CPMV-HT/NOS expression cassette usingIn-Fusion cloning system (Clontech, Mountain View, Calif.). Constructnumber 1191 (FIG. 7D) was digested with SacII and StuI restrictionenzyme and the linearized plasmid was used for the In-Fusion assemblyreaction. Construct number 1191 is an acceptor plasmid intended for “InFusion” cloning of genes of interest in a CPMV-HT-based expressioncassette. It also incorporates a gene construct for the co-expression ofthe TBSV P19 suppressor of silencing under the alfalfa Plastocyanin genepromoter and terminator. The backbone is a pCAMBIA binary plasmid andthe sequence from left to right t-DNA borders is presented in FIG. 7E(SEQ ID NO: 22). The resulting construct was given number 1730 (FIG. 9D,SEQ ID NO: 33). The amino acid sequence of VP4 from Rotavirus A vaccineUSA/Rotarix-A41CB052A/1988/G1P1A[8] is presented in FIG. 9E (SEQ ID NO:34). A representation of plasmid 1730 is presented in FIG. 9F.

4. 2X35S/CPMV-HT/TrSp-RVA(Rtx) VP7(opt)/NOS (Construct Number 1734)

An optimized sequence encoding VP7 with a truncated version of thenative signal peptide from Rotavirus A vaccineUSA/Rotarix-A41CB052A/1988/G1P1A[8] strain was cloned into2X35S-CPMV-HT-NOS expression system in a plasmid containingPlasto_pro/P19/Plasto_ter expression cassette using the followingPCR-based method. A fragment containing the VP7 coding sequence wasamplified using primers IF-TrSP+Rtx_VP7(opt).s1+3c (FIG. 10A, SEQ ID NO:35) and IF-Rtx_VP7(opt).s1−4r (FIG. 10B, SEQ ID NO: 36), using optimizedVP7 gene sequence (corresponding to nt 88-891 from FIG. 10C, SEQ ID NO:37) as template. For sequence optimization, VP7 protein sequence(Genbank accession number AEX30682) was backtranslated and optimized forhuman codon usage, GC content and mRNA structure. The PCR product wascloned in 2X35S/CPMV-HT/NOS expression system using In-Fusion cloningsystem (Clontech, Mountain View, Calif.). Construct number 1191 (FIG.7D) was digested with SacII and StuI restriction enzyme and thelinearized plasmid was used for the In-Fusion assembly reaction.Construct number 1191 is an acceptor plasmid intended for “In Fusion”cloning of genes of interest in a CPMV-HT-based expression cassette. Italso incorporates a gene construct for the co-expression of the TBSV P19suppressor of silencing under the alfalfa Plastocyanin gene promoter andterminator. The backbone is a pCAMBIA binary plasmid and the sequencefrom left to right t-DNA borders is presented in FIG. 7E (SEQ ID NO:22). The resulting construct was given number 1734 (FIG. 10D, SEQ ID NO:38). The amino acid sequence of VP7 with truncated signal peptide fromRotavirus A vaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] strain ispresented in FIG. 10E (SEQ ID NO: 39). A representation of plasmid 1734is presented in FIG. 10F.

5. 2X35S/CPMV-HT/RVA(WA) NSP4/NOS (Construct Number 1706)

A sequence encoding NSP4 from Rotavirus A WA strain was cloned into2X35S-CPMV-HT-NOS expression system in a plasmid containingPlasto_pro/P19/Plasto_ter expression cassette using the followingPCR-based method. A fragment containing the NSP4 coding sequence wasamplified using primers IF-WA_NSP4.s1+3c (FIG. 11A, SEQ ID NO: 40) andIF-WA_NSP4.s1−4r (FIG. 11B, SEQ ID NO: 41), using synthesized NSP4 gene(corresponding to nt 42-569 from GenBank accession number K02032) (FIG.11C, SEQ ID NO: 42) as template. The PCR product was cloned in2X35S/CPMV-HT/NOS expression system using In-Fusion cloning system(Clontech, Mountain View, Calif.). Construct number 1191 (FIG. 7D) wasdigested with SacII and StuI restriction enzyme and the linearizedplasmid was used for the In-Fusion assembly reaction. Construct number1191 is an acceptor plasmid intended for “In Fusion” cloning of genes ofinterest in a CPMV-HT-based expression cassette. It also incorporates agene construct for the co-expression of the TBSV P19 suppressor ofsilencing under the alfalfa Plastocyanin gene promoter and terminator.The backbone is a pCAMBIA binary plasmid and the sequence from left toright t-DNA borders is presented in FIG. 7E (SEQ ID NO: 22). Theresulting construct was given number 1706 (FIG. 11D, SEQ ID NO: 43). Theamino acid sequence of NSP4 from Rotavirus A strain WA is presented inFIG. 11E (SEQ ID NO: 44). A representation of plasmid 1706 is presentedin FIG. 11F.

6. 2X35S/CPMV-160/RVA(WA) VP2(opt)/NOS (Construct Number 1108)

An optimized sequence encoding VP2 from Rotavirus A WA strain was clonedinto 2X35S/CPMV-160/NOS expression system in a plasmid containingPlasto_pro/P19/Plasto_ter expression cassette using the followingPCR-based method. A fragment containing the VP2 coding sequence wasamplified using primers IF(C160)-WA_VP2(opt).c (FIG. 12A, SEQ ID NO: 45)and IF-WA_VP2(opt).s1−4r (FIG. 7B, SEQ ID NO: 20), using optimized VP2gene sequence (FIG. 7C, SEQ ID NO: 21) as template. For sequenceoptimization, VP2 protein sequence (Genbank accession number CAA33074)was backtranslated and optimized for human codon usage, GC content andmRNA structure. The PCR product was cloned in 2X35S/CPMV-160/NOSexpression system using In-Fusion cloning system (Clontech, MountainView, Calif.). Construct number 1190 (FIG. 12B) was digested with SacIIand StuI restriction enzyme and the linearized plasmid was used for theIn-Fusion assembly reaction. Construct number 1190 is an acceptorplasmid intended for “In Fusion” cloning of genes of interest in aCPMV-160-based expression cassette. It also incorporates a geneconstruct for the co-expression of the TBSV P19 suppressor of silencingunder the alfalfa Plastocyanin gene promoter and terminator. Thebackbone is a pCAMBIA binary plasmid and the sequence from left to rightt-DNA borders is presented in FIG. 12C (SEQ ID NO: 46). The resultingconstruct was given number 1108 (FIG. 12D, SEQ ID NO: 47). The aminoacid sequence of VP2 from Rotavirus A strain WA is presented in FIG. 7G(SEQ ID NO: 24). A representation of plasmid 1108 is presented in FIG.12E.

7.—2X35S/CPMV-160/RVA(WA) VP6(opt)/NOS (Construct Number 1128)—

An optimized sequence encoding VP6 from Rotavirus A WA strain was clonedinto 2X35S/CPMV-160/NOS expression system in a plasmid containingPlasto_pro/P19/Plasto_ter expression cassette using the followingPCR-based method. A fragment containing the VP6 coding sequence wasamplified using primers IF(C160)-WA_VP6(opt).c (FIG. 13A, SEQ ID NO: 48)and IF-WA_VP6(opt).s1−4r (FIG. 8B, SEQ ID NO: 26), using optimized VP6gene sequence (FIG. 8C, SEQ ID NO: 27) as template. For sequenceoptimization, VP6 protein sequence (Genbank accession number AAA47311)was backtranslated and optimized for human codon usage, GC content andmRNA structure. The PCR product was cloned in 2X35S/CPMV-160/NOSexpression system using In-Fusion cloning system (Clontech, MountainView, Calif.). Construct number 1190 (FIG. 11B, SEQ ID NO:40) wasdigested with SacII and StuI restriction enzyme and the linearizedplasmid was used for the In-Fusion assembly reaction. Construct number1190 is an acceptor plasmid intended for “In Fusion” cloning of genes ofinterest in a CPMV-160-based expression cassette. It also incorporates agene construct for the co-expression of the TBSV P19 suppressor ofsilencing under the alfalfa Plastocyanin gene promoter and terminator.The backbone is a pCAMBIA binary plasmid and the sequence from left toright t-DNA borders is presented in FIG. 11C (SEQ ID NO: 41). Theresulting construct was given number 1128 (FIG. 13B, SEQ ID NO: 49). Theamino acid sequence of VP6 from Rotavirus A strain WA is presented inFIG. 8E (SEQ ID NO: 28). A representation of plasmid 1128 is presentedin FIG. 13C.

8. X35S/CPMV-160/RVA(Rtx) VP4(opt)/NOS (Construct Number 1178)

An optimized sequence encoding VP4 from Rotavirus A vaccineUSA/Rotarix-A41CB052A/1988/G1P1A[8] strain was cloned into2X35S/CPMV-160/NOS in a plasmid containing Plasto_pro/P19/Plasto_terexpression cassette using the following PCR-based method. A fragmentcontaining the VP4 coding sequence was amplified using primersIF(C160)-Rtx_VP4(opt).c (FIG. 14A, SEQ ID NO: 50) andIF-Rtx_VP4(opt).s1−4r (FIG. 9B, SEQ ID NO: 30), using optimized VP4 genesequence (FIG. 9C, SEQ ID NO: 31) as template. For sequenceoptimization, VP4 protein sequence (Genbank accession number AEX30660)was backtranslated and optimized for human codon usage, GC content andmRNA structure. The PCR product was cloned in 2X35S/CPMV-160/NOSexpression system using In-Fusion cloning system (Clontech, MountainView, Calif.). Construct number 1190 (FIG. 11B, SEQ ID NO: 40) wasdigested with SacII and StuI restriction enzyme and the linearizedplasmid was used for the In-Fusion assembly reaction. Construct number1190 is an acceptor plasmid intended for “In Fusion” cloning of genes ofinterest in a CPMV-160-based expression cassette. It also incorporates agene construct for the co-expression of the TBSV P19 suppressor ofsilencing under the alfalfa Plastocyanin gene promoter and terminator.The backbone is a pCAMBIA binary plasmid and the sequence from left toright t-DNA borders is presented in FIG. 11C (SEQ ID NO: 41). Theresulting construct was given number 1178 (FIG. H2, SEQ ID NO: H2). Theamino acid sequence of VP4 from Rotavirus A vaccineUSA/Rotarix-A41CB052A/1988/G1P1A[8] is presented in FIG. 9E (SEQ ID NO:33). A representation of plasmid 1178 is presented in FIG. 14C.

9. 2X35S/CPMV-160/TrSp-RVA(Rtx) VP7(opt)/NOS (Construct Number 1199)

An optimized sequence encoding VP7 with a truncated version of thenative signal peptide from Rotavirus A vaccineUSA/Rotarix-A41CB052A/1988/G1P1A[8] strain was cloned into2X35S/CPMV-160/NOS expression system in a plasmid containingPlasto_pro/P19/Plasto_ter expression cassette using the followingPCR-based method. A fragment containing the VP7 coding sequence wasamplified using primers IF(C160)-TrSP+Rtx_VP7(opt).c (FIG. 15A, SEQ IDNO: 52) and IF-Rtx_VP7(opt).s1−4r (FIG. 10B, SEQ ID NO: 35), usingoptimized VP7 gene sequence (corresponding to nt 88-891 from FIG. 10C,SEQ ID NO: 36) as template. For sequence optimization, VP7 proteinsequence (Genbank accession number AEX30682) was backtranslated andoptimized for human codon usage, GC content and mRNA structure. The PCRproduct was cloned in 2X35S/CPMV-160/NOS expression system usingIn-Fusion cloning system (Clontech, Mountain View, Calif.). Constructnumber 1190 (FIG. 11B, SEQ ID NO: 40) was digested with SacII and StuIrestriction enzyme and the linearized plasmid was used for the In-Fusionassembly reaction. Construct number 1190 is an acceptor plasmid intendedfor “In Fusion” cloning of genes of interest in a CPMV-160-basedexpression cassette. It also incorporates a gene construct for theco-expression of the TBSV P19 suppressor of silencing under the alfalfaPlastocyanin gene promoter and terminator. The backbone is a pCAMBIAbinary plasmid and the sequence from left to right t-DNA borders ispresented in FIG. 11C (SEQ ID NO: 41). The resulting construct was givennumber 1199 (FIG. 15B, SEQ ID NO: 53). The amino acid sequence of VP7with truncated signal peptide from Rotavirus A vaccineUSA/Rotarix-A41CB052A/1988/G1P1A[8] strain is presented in FIG. 10E (SEQID NO: 38). A representation of plasmid 1199 is presented in FIG. 15C.

10. Double Gene Construct for the Expression of VP6 and VP2 UnderCPMV-HT Expression Cassette (Construct Number 1708)

A single vector for the co-expression of VP6 from Rotavirus A WA strainand VP2 from Rotavirus A WA strain under the control of CPMV-HTexpression system was assembled using the following restrictionenzyme/ligase-based method. Donor plasmid DNA (construct number 1710;2X35S/CPMV-HT/RVA(WA) VP2(opt)/NOS) (FIG. 7F, SEQ ID NO: 23) wasdigested with AvrII (located before the 2X35S promoter) and AscI(located after the NOS terminator) restriction enzymes and the fragmentcorresponding to 2X35S/CPMV-HT/RVA(WA) VP2(opt)/NOS expression cassettewas gel-purified. This fragment was then inserted into the acceptorconstruct number 1713 (2X35S/CPMV-HT/RVA(WA) VP6(opt)/NOS) (FIG. 8D, SEQID NO: 28) linearized using XbaI and AscI restriction enzymes (bothsites are located after the NOS terminator of VP6 expression cassette).The resulting construct was given number 1708. A representation ofplasmid 1708 is presented in FIG. 16.

11. Double Gene Construct for the Expression of VP7 and VP4 UnderCPMV-HT Expression Cassette (Construct Number 1719)

A single vector for the co-expression of VP7 with a truncated version ofthe native signal peptide from Rotavirus A vaccineUSA/Rotarix-A41CB052A/1988/G1P1A[8] strain and VP4 from Rotavirus Avaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] strain under the control ofCPMV-HT expression system was assembled using the following restrictionenzyme/ligase-based method. Donor plasmid DNA (construct number 1730;2X35S/CPMV-HT/RVA(Rtx) VP4(opt)/NOS) (FIG. 9D, SEQ ID NO: 32) wasdigested with AvrII (located before the 2X35S promoter) and AscI(located after the NOS terminator) restriction enzymes and the fragmentcorresponding to 2X35S/CPMV-HT/RVA(Rtx) VP4(opt)/NOS expression cassettewas gel-purified. This fragment was then inserted into the acceptorconstruct number 1734 (2X35S/CPMV-HT/TrSp-RVA(Rtx) VP7(opt)/NOS) (FIG.10D, SEQ ID NO: 37) linearized using XbaI and AscI restriction enzymes(both sites are located after the NOS terminator of VP7 expressioncassette). The resulting construct was given number 1719. Arepresentation of plasmid 1719 is presented in FIG. 17.

12. Double Gene Construct for the Expression of VP6 and VP2 UnderCPMV-160 Expression Cassette (Construct Number 2400)

A single vector for the co-expression of VP6 from Rotavirus A WA strainand VP2 from Rotavirus A WA strain under the control of CPMV-160expression system was assembled using the following restrictionenzyme/ligase-based method. Donor plasmid DNA (construct number 1108;2X35S/CPMV-160/RVA(WA) VP2(opt)/NOS) (FIG. 12D, SEQ ID NO: 47) wasdigested with AvrII (located before the 2X35S promoter) and AscI(located after the NOS terminator) restriction enzymes and the fragmentcorresponding to 2X35S/CPMV-160/RVA(WA) VP2(opt)/NOS expression cassettewas gel-purified. This fragment was then inserted into the acceptorconstruct number 1128 (2X35S/CPMV-160/RVA(WA) VP6(opt)/NOS) (FIG. 13B,SEQ ID NO: 49) linearized using XbaI and AscI restriction enzymes (bothsites are located after the NOS terminator of VP6 expression cassette).The resulting construct was given number 2400. A representation ofplasmid 2400 is presented in FIG. 18.

13. Double Gene Construct for the Expression of VP7 and VP4 UnderCPMV-160 Expression Cassette (Construct Number 2408)

A single vector for the co-expression of VP7 with a truncated version ofthe native signal peptide from Rotavirus A vaccineUSA/Rotarix-A41CB052A/1988/G1P1A[8] strain and VP4 from Rotavirus Avaccine USA/Rotarix-A41CB052A/1988/G1P1A[8] strain under the control ofCPMV-160 expression system was assembled using the following restrictionenzyme/ligase-based method. Donor plasmid DNA (construct number 1178;2X35S/CPMV-160/RVA(Rtx) VP4(opt)/NOS) (FIG. 14B, SEQ ID NO: 51) wasdigested with AvrII (located before the 2X35S promoter) and AscI(located after the NOS terminator) restriction enzymes and the fragmentcorresponding to 2X35S/CPMV-160/RVA(Rtx) VP4(opt)/NOS expressioncassette was gel-purified. This fragment was then inserted into theacceptor construct number 1199 (2X35S/CPMV-160/TrSp-RVA(Rtx)VP7(opt)/NOS) (FIG. 15B, SEQ ID NO: 53) linearized using XbaI and AscIrestriction enzymes (both sites are located after the NOS terminator ofVP7 expression cassette). The resulting construct was given number 2408.A representation of plasmid 2408 is presented in FIG. 19.

14. Quadruple Gene Construct for the Expression of VP7, VP4, VP6 and VP2Under CPMV-HT Expression Cassette (Construct Number 1769)

A single vector for the co-expression of VP7 with a truncated version ofthe native signal peptide from Rotavirus A vaccineUSA/Rotarix-A41CB052A/1988/G1P1A[8] strain, VP4 from Rotavirus A vaccineUSA/Rotarix-A41CB052A/1988/G1P1A[8] strain, VP6 from Rotavirus A WAstrain and VP2 from Rotavirus A WA strain under the control of CPMV-HTexpression system was assembled using the following restrictionenzyme/ligase-based method. Donor plasmid DNA (construct number 1730;2X35S/CPMV-HT/RVA(Rtx) VP4(opt)/NOS) (FIG. 9D, SEQ ID NO: 32) wasdigested with AvrII (located before the 2X35S promoter) and AscI(located after the NOS terminator) restriction enzymes and the fragmentcorresponding to 2X35S/CPMV-HT/RVA(Rtx) VP4(opt)/NOS expression cassettewas gel-purified. This fragment was then inserted into the acceptorconstruct number 1734 (2X35S/CPMV-HT/TrSp-RVA(Rtx) VP7(opt)/NOS) (FIG.10D, SEQ ID NO: 37) linearized using XbaI and AscI restriction enzymes(both sites are located after the NOS terminator of VP7 expressioncassette). Ligation of cohesive ends produced by AvrII and XbaIdestroyed the original restriction sites producing a temporary acceptorvector with the same unique XbaI and AscI restriction enzyme sites atthe end of the NOS terminator of the second expression cassettes (fromleft to right T-DNA). VP6 (construct number 1713; FIG. 8D, SEQ ID NO:28) and VP2 (construct number 1710; FIG. 7F, SEQ ID NO: 23) expressedunder CPMV-HT expression system were then inserted sequentially in theresulting temporary acceptor vector using the same digestion strategy togive the final VP7/VP4/VP6/VP2 construct. The resulting construct wasgiven number 1769. A representation of plasmid 1769 is presented in FIG.20.

15. Quintuple Gene Construct for the Expression of VP4, VP7, NSP4, VP6and VP2 Under CPMV-HT Expression Cassette (Construct Number 2441)

A single vector for the co-expression of VP4 from Rotavirus A vaccineUSA/Rotarix-A41CB052A/1988/G1P1A[8] strain, VP7 with a truncated versionof the native signal peptide from Rotavirus A vaccineUSA/Rotarix-A41CB052A/1988/G1P1A[8] strain, NSP4 from Rotavirus A WAstrain, VP6 from Rotavirus A WA strain and VP2 from Rotavirus A WAstrain under the control of CPMV-HT expression system was assembledusing the following restriction enzyme/ligase-based method. Donorplasmid DNA (construct number 1734; 2X35S/CPMV-HT/TrSp-RVA(Rtx)VP7(opt)/NOS) (FIG. 10D, SEQ ID NO: 37) was digested with AvrII (locatedbefore the 2X35S promoter) and AscI (located after the NOS terminator)restriction enzymes and the fragment corresponding to2X35S/CPMV-HT/TrSp-RVA(Rtx) VP7(opt)/NOS expression cassette wasgel-purified. This fragment was then inserted into the acceptorconstruct number 1730 (2X35S/CPMV-HT/RVA(Rtx) VP4(opt)/NOS) (FIG. 9D,SEQ ID NO: 32) linearized using XbaI and AscI restriction enzymes (bothsites are located after the NOS terminator of VP4 expression cassette).Ligation of cohesive ends produced by AvrII and XbaI destroyed theoriginal restriction sites producing a temporary acceptor vector withthe same unique XbaI and AscI restriction enzyme sites at the end of theNOS terminator of the second expression cassettes (from left to rightT-DNA). NSP4 (construct number 1706; FIG. 11D, SEQ ID NO: 42), VP6(construct number 1713; FIG. 8D, SEQ ID NO: 28) and VP2 (constructnumber 1710; FIG. 7F, SEQ ID NO: 23) expressed under CPMV-HT expressionsystem were then inserted sequentially in the resulting temporaryacceptor vector using the same digestion strategy to give the finalVP4/VP7/NSP4/VP6/VP2 construct. The resulting construct was given number2441. A representation of plasmid 2441 is presented in FIG. 21.

Example 2 Co-Expression of NSP4 Increases VP4 and VP4 Incorporation intoRLPs

The rotavirus VP2, VP4, VP6 and VP7 structural antigens were transientlyco-expressed in Nicotiana benthamiana plants in the presence or absenceof a NSP4 expression construct using agroinfiltration as described inexample 1. Crude protein extracts from RLP producing plants containlarge amounts of host protein as shown by the banding pattern inCoomassie-stained SDS-PAGE (FIG. 2B, load). Rotavirus-like particles canbe separated from plant proteins by ultracentrifugation on a iodixanoldensity gradient. After centrifugation, analysis of the fractions fromiodixanol density gradient showed that the RLPs migrated to the 35%iodixanol fraction (F2 and F3 in FIG. 2B) while the majority of the hostproteins remained in the 25-30% iodixanol fractions (F4-F10 in FIG. 2B).RLPs from plants co-expressing rotavirus structural antigens werepurified on iodixanol density gradients and the analysis of the RLPcontaining fractions (F2 and F3) showed that RLPs can be producedefficiently, irrespectively of the number of gene per construct as shownin FIG. 3A with single, dual and quadruple gene constructs. The resultsobtained also showed that the co-expression of NSP4 reduced RLPexpression (compare fractions under −NSP4 and +NSP4 in FIG. 3A). Notethat equal volumes of each fraction were loaded on the gel to compareRLP content per volume.

RLP-containing fraction 2 from the same experiments were analyzed bywestern blot to evaluate the impact of NSP4 co-expression on VP4 and VP7incorporation. For that comparison, equal amounts of RLPS were loaded onthe gel. The western blot results obtained showed stronger signals forVP4 and VP7 on the RLPs produced in the presence of NSP4 (FIG. 3B,compare lanes under −NSP4 and +NSP4). These results clearly indicatethat the co-expression of NSP4 increased VP4 and VP7 incorporation onthe surface of the RLPs.

The genes encoding the four rotavirus antigens and the non-structuralprotein NSP4 were cloned into CPMV-HT and CPMV160 for comparison ofexpression. Co-expression studies followed by extraction andpurification by ultracentrifugation in iodixanol density gradient showedthat both expression efficiently produced RLPs, as demonstrated by theamount of VP6 in fractions 2 and 3 of the gradient (FIG. 4A), and theamount of VP4 and VP7 in fraction 2 from the same treatments (FIG. 4B).This study also showed that, when using the CPMV-HT system forexpression of the rotavirus proteins, single gene constructs produced asmuch RLPs as dual gene constructs (FIG. 4A, left panel vs middle panel)and resulted in similar coverage with the surface antigens, VP4 and VP7(FIG. 4B, left panel vs middle panel).

A quintuple gene construct (comprising 5 genes on the same plasmid) hasbeen evaluated for the co-expression of the four structural antigenswith NSP4. As shown in FIG. 5, the use of quintuple gene constructresulted in similar RLP production level as with the use of a quadruplegene construct with the NSP4 gene on a separate plasmid (FIG. 5, toppanel), as well as comparable levels of VP4 and VP7 incorporation (FIG.5, lower panel).

Agrobacterium Transformation

All plasmids were used to transform Agrobacterium tumefaciens (AGL1;ATCC, Manassas, Va. 20108, USA) by electroporation (Mattanovich et al.,1989, Nucleic Acid Res. 17:6747) alternatively, heat shock usingCaCl2-prepared competent cells (XU et al., 2008, Plant Methods 4) may beused. The integrity of the plasmids in the A. tumefaciens strainscreated was confirmed by restriction mapping.

Preparation of Plant Biomass, Inoculum, Agroinfiltration, and Harvesting

Nicotiana benthamiana plants were grown from seeds in flats filled witha commercial peat moss substrate. The plants were allowed to grow in thegreenhouse under a 16/8 photoperiod and a temperature regime of 25° C.day/20° C. night. Three weeks after seeding, individual plantlets werepicked out, transplanted in pots and left to grow in the greenhouse forthree additional weeks under the same environmental conditions.

Agrobacteria transfected with each construct were grown in a LB mediumfrom vegetal origin and supplemented with 10 mM2-(N-morpholino)ethanesulfonic acid (MES) and 50 μg/ml kanamycin pH5.6until they reached an OD600 between 0.6 and 2.5. Agrobacteriumsuspensions were mixed to reach appropriate ratio for each construct andbrought to 2.5× OD600 with infiltration medium (10 mM MgCl2 and 10 mMMES pH 5.6). A. tumefaciens suspensions were stored overnight at 4° C.On the day of infiltration, culture batches were diluted withinfiltration medium and allowed to warm before use. Whole plants of N.benthamiana were placed upside down in the bacterial suspension in anair-tight stainless steel tank under a vacuum of 20-40 Torr for 2-min.Following infiltration, plants were returned to the greenhouse for a 9day incubation period until harvest. Harvested biomass was kept frozen(−80° C.) until use for purification of particles.

Extraction and Screening by Ultracentrifugation of Rotavirus-LikeParticles

Proteins were extracted from frozen biomass by mechanical extraction ina blender with 2 volumes of extraction buffer (TNC: 10 mM Tris pH 7.4,140 mM NaCl, 10 mM CaCl2). The slurry was filtered through a large porenylon filter to remove large debris and centrifuged 5000 g for 5 min at4° C. The supernatant was collected and centrifuged again at 5000 g for30 min (4° C.) to remove additional debris. The supernatant is thenloaded on a discontinuous iodixanol density gradient.

Analytical density gradient centrifugation was performed as follows. 38ml tubes containing discontinuous iodixanol density gradient in TNCbuffer (1.2 ml at 45%, 2 ml at 35%, 5 ml at 30% and 5 ml at 25% ofiodixanol) were prepared and overlaid with 25 ml of the extractscontaining the rotavirus-like particles. The gradients were centrifugedat 120 000 g for 4 hours (4° C.). After centrifugation, 1 ml fractionswere collected from the bottom to the top and fractions 2 and 3(corresponding to 35% iodixanol) were analysed by SDS-PAGE combined toprotein staining or Western blot.

SDS-PAGE and Immunoblotting

Protein concentrations were determined by the BCA protein assay (PierceBiochemicals, Rockport, Ill.). Proteins were separated by SDS-PAGE underreducing conditions using Criterion™ TGX Stain-Free™ precast gels(Bio-Rad Laboratories, Hercules, Calif.) and proteins were visualizedwith Gel Doc™ EZ imaging system (Bio-Rad Laboratories, Hercules,Calif.).

For immunoblotting, electrophoresed proteins were electrotransferredonto polyvinylene difluoride (PVDF) membranes (Roche DiagnosticsCorporation, Indianapolis, Ind.). Prior to immunoblotting, the membraneswere blocked with 5% skim milk and 0.1% Tween-20 in Tris-buffered saline(TBS-T) for 16-18 h at 4° C.

Immunoblotting was performed by incubation with a suitable antibody(Table 4) in 2% skim milk in TBS-Tween 20 0.1%. Secondary antibodiesused for chemiluminescence detection were as indicated in Table 4,diluted as indicated in 2% skim milk in TBS-Tween 20 0.1% Immunoreactivecomplexes were detected by chemiluminescence using luminol as thesubstrate (Roche Diagnostics Corporation, Indianapolis, Ind.).

TABLE 4 Electrophoresis conditions, antibodies, and dilutions forimmunoblotting of rotavirus antigens. Rota- Electro- virus phoresisanti- condi- Secondary gen tion Primary antibody Dilution antibodyDilution VP4 Re- Rabbit serum from 1:30 000 Goat anti- 1:10 000 ducingimmunized Rabbit rabbit (JIR with recombinant 111-035- VP4 (in house)144) VP7 Re- Rabbit serum from 1:50 000 Goat anti- 1:10 000 ducingimmunized Rabbit rabbit (JIR with recombinant 111-035- VP7 (in house)144)

All citations are hereby incorporated by reference.

The present invention has been described with regard to one or moreembodiments. However, it will be apparent to persons skilled in the artthat a number of variations and modifications can be made withoutdeparting from the scope of the invention as defined in the claims.

What is claimed is:
 1. A method of producing a rotavirus like particle(RLP) in a host or host cell comprising: a) providing a host or hostcell comprising one or more nucleic acid comprising a first nucleotidesequence encoding a first rotavirus protein, a second nucleotidesequence encoding a second rotavirus protein and a third nucleotidesequence encoding a third rotavirus protein, the first, second and thirdnucleotide sequence being operatively linked to one or more regulatoryregion active in the host or host cell; and the first nucleotidesequence encoding rotavirus protein NSP4, the second nucleotide sequenceencoding rotavirus protein VP6, and the third nucleotide sequenceencoding one of rotavirus protein VP7 or VP4; b) incubating the host orhost cell under conditions that permit the expression of the one or morenucleic acid, so that each of NSP4, VP6 and VP7 or VP4 are expressed,thereby producing the RLP, the RLP comprising rotavirus proteins, therotavirus proteins consisting of rotavirus structural proteins.
 2. Themethod of claim 1, wherein the one or more nucleic acid furthercomprises a fourth nucleotide sequence encoding a fourth rotavirusprotein, the first, second, third, and fourth nucleotide sequence beingoperatively linked to one or more regulatory region active in the hostor host cell; and the first nucleotide sequence encoding rotavirusprotein NSP4, the second nucleotide sequence encoding rotavirus proteinVP6, and the third and fourth nucleotide sequence encoding rotavirusprotein VP2, VP4 or VP7 and wherein each of NSP4, VP6 and two of VP2,VP4 and VP7 are expressed from the one or more nucleic acid.
 3. Themethod of claim 2, wherein the one or more nucleic acid furthercomprises a fifth nucleotide sequence encoding a fifth rotavirusprotein, the first, second, third, fourth and fifth nucleotide sequencebeing operatively linked to one or more regulatory region active in thehost or host cell; and the first, second, third, fourth and fifthnucleotide sequence encoding one of rotavirus protein VP2, VP4, VP6, VP7or NSP4 and wherein each of VP2, VP4, VP6, VP7 and NSP4 are expressedfrom the one or more nucleic acid.
 4. The method of claim 1, wherein thehost or host cell comprises insect cells, mammalian cells, plant,portion of a plant or plant cells.
 5. The method of claim 1, wherein theone or more nucleotide sequence is operatively linked to one or moreexpression enhancer.
 6. The method of claim 5, wherein the expressionenhancer is selected from the group consisting of CPMV HT, CPMV 160,CPMV 160+ and CPMV HT+.
 7. The method of claim 1, further comprising thesteps of: c) harvesting the host or host cell, and d) purifying the RLPsfrom the host or host cell, wherein the RLPs range in size from 70-100nm.
 8. A method of producing a rotavirus like particle (RLP) in a hostor host cell comprising: a) introducing into the host or host cell oneor more nucleic acid comprising a first nucleotide sequence encoding afirst rotavirus protein, a second nucleotide sequence encoding a secondrotavirus protein and a third nucleotide sequence encoding a thirdrotavirus protein, the first, second and third nucleotide sequence beingoperatively linked to one or more regulatory region active in the hostor host cell; and the first nucleotide sequence encoding rotavirusprotein NSP4, the second nucleotide sequence encoding rotavirus proteinVP6, and the third nucleotide sequence encoding one of rotavirus proteinVP7 or VP4; b) incubating the host or host cell under conditions thatpermit the expression of the one or more nucleic acid so that each ofNSP4, VP6 and VP7 or VP4 are expressed, thereby producing the RLP, theRLP comprising rotavirus proteins, the rotavirus proteins consisting ofrotavirus structural proteins.
 9. The method of claim 8, wherein the oneor more nucleic acid further comprises a fourth nucleotide sequenceencoding a fourth rotavirus protein, the first, second, third, andfourth nucleotide sequence being operatively linked to one or moreregulatory region active in the host or host cell; and the firstnucleotide sequence encoding rotavirus protein NSP4, the secondnucleotide sequence encoding rotavirus protein VP6, and the third andfourth nucleotide sequence encoding of rotavirus protein VP2, VP4 or VP7and wherein each of NSP4, VP6 and two of VP2, VP4 and VP7 are expressedfrom the one or more nucleic acid.
 10. The method of claim 9, whereinthe one or more nucleic acid further comprises a fifth nucleotidesequence encoding a fifth rotavirus protein, the first, second, third,fourth and fifth nucleotide sequence being operatively linked to one ormore regulatory region active in the host or host cell; and the first,second, third, fourth and fifth nucleotide sequence encoding one ofrotavirus protein VP2, VP4, VP6, VP7 or NSP4 and wherein each of VP2,VP4, VP6, VP7 and NSP4 are expressed from the one or more nucleic acid.11. The method of claim 8, wherein the one or more nucleotide sequenceis operatively linked to one or more expression enhancer.
 12. The methodof claim 11, wherein the expression enhancer is selected from the groupconsisting of CPMV HT, CPMV 160, CPMV 160+ and CPMV HT+.
 13. The methodof claim 8, further comprising the steps of: c) harvesting the host orhost cell, and d) purifying the RLPs from the host or host cell, whereinthe RLPs range in size from 70-100 nm.
 14. An RLP produced by the methodof claim 8, wherein the RLP is a triple layered RLP comprising rotavirusprotein, the rotavirus protein consisting of VP2, VP4, VP6 and VP7. 15.A composition comprising an effective dose of the RLP of claim 14 forinducing an immune response in a subject, and a pharmaceuticallyacceptable carrier.
 16. A method of inducing immunity to a rotavirusinfection in a subject, comprising administering the composition ofclaim 15 to the subject.
 17. The method of claim 16, wherein thecomposition is administered to a subject orally, intradermally,intranasally, intramuscularly, intraperitoneally, intravenously, orsubcutaneously.
 18. The method of claim 8, wherein the first rotavirusprotein, the second rotavirus protein and the third rotavirus proteinare obtained from any one rotavirus strain having a genotype G1 to G27.19. The method of claim 8, wherein the first rotavirus protein, thesecond rotavirus protein and the third rotavirus protein are obtainedfrom a rotavirus strain having a genotype G1 or G4.
 20. A plant, portionof a plant, a plant cell, a plant matter, or a plant extract comprisingan RLP produced by the method of claim
 8. 21. The method of claim 1,wherein the host or host cell comprises plant, portion of plant or plantcells.
 22. The method of claim 2, wherein the third and fourthnucleotide sequence encoding rotavirus protein VP2 or VP7 and whereineach of VP2, VP6, VP7 and NSP4 are expressed from the one or morenucleic acid.
 23. The method of claim 22, wherein the host or host cellcomprises Nicotiana benthamiana, portion of Nicotiana benthamiana orNicotiana benthamiana cells.
 24. The method of claim 23, wherein thenucleotide sequence encoding VP2 comprises from 80% to 100% identitywith a nucleotide sequence as defined by SEQ ID NO: 21, the nucleotidesequence encoding VP6 comprises from 80% to 100% identity with anucleotide sequence as defined by SEQ ID NO:27, the nucleotide sequenceencoding VP7 comprises from 80% to 100% identity with a nucleotidesequence as defined by SEQ ID NO:37, the nucleotide sequence encodingNSP4 comprises from 80% to 100% identity with a nucleotide sequence asdefined by SEQ ID NO:42.
 25. The method of claim 23, wherein the VP2comprises an amino acid sequence having from 80% to 100% identity withthe amino acid sequence as defined by SEQ ID NO:24, the VP6 comprises anamino acid sequence having from 80% to 100% identity with the amino acidsequence as defined by SEQ ID NO:29, the VP7 comprises an amino acidsequence having from 80% to 100% identity with the amino acid sequenceas defined by SEQ ID NO: 39, the NSP4 comprises an amino acid sequencehaving from 80% to 100% identity with the amino acid sequence as definedby SEQ ID NO:44.